Cancer immunotherapy by disrupting pd-1/pd-l1 signaling

ABSTRACT

The disclosure provides a method for immunotherapy of a subject afflicted with cancer, comprises administering to the subject a composition comprising a therapeutically effective amount of an antibody that inhibits signaling from the PD-1/PD-L1 signaling pathway. This disclosure also provides a method for immunotherapy of a subject afflicted with cancer comprising selecting a subject that is a suitable candidate for immunotherapy based on an assessment that the proportion of cells in a test tissue sample from the subject that express PD-L1 on the cell surface exceeds a predetermined threshold level, and administering a therapeutically effective amount of an anti-PD-1 antibody to the selected subject. The invention additionally provides rabbit mAbs that bind specifically to a cell surface-expressed PD-L1 antigen in a FFPE tissue sample, and an automated IHC method for assessing cell surface expression in FFPE tissues using the provided anti-PD-L1 Abs.

Throughout this application, various publications are referenced inparentheses by author name and date, or by Patent No. or PatentPublication No. Full citations for these publications may be found atthe end of the specification immediately preceding the claims. Thedisclosures of these publications are hereby incorporated in theirentireties by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein. However, thecitation of a reference herein should not be construed as anacknowledgement that such reference is prior art to the presentinvention.

FIELD OF THE INVENTION

This invention relates to methods for immunotherapy of a cancer patientcomprising administering to the patient antibodies that disrupt thePD-1/PD-L1 signaling pathway. A biomarker may be used as part of thistreatment for identifying suitable patients for immunotherapy and forpredicting the efficacy of anti-PD-1 treatment.

BACKGROUND OF THE INVENTION

Human cancers harbor numerous genetic and epigenetic alterations,generating neoantigens potentially recognizable by the immune system(Sjoblom et al., 2006). Although an endogenous immune response to canceris observed in preclinical models and patients, this response isineffective, and established cancers are viewed as “self” and toleratedby the immune system. Contributing to this state of tolerance, tumorsmay exploit several distinct mechanisms to actively suppress the hostimmune response (Topalian et al., 2011; Mellman et al., 2011). Amongthese mechanisms, endogenous “immune checkpoints” that normallyterminate immune responses to mitigate collateral tissue damage can beco-opted by tumors to evade immune destruction. Intensive efforts todevelop specific immune checkpoint pathway inhibitors have begun toprovide new immunotherapeutic approaches for treating cancer, includingthe development of the anti-CTLA-4 antibody (Ab), ipilimumab (YERVOY®),for the treatment of patients with advanced melanoma (Hodi et al.,2010).

Programmed Death-1 (PD-1) is a key immune checkpoint receptor expressedby activated T and B cells and mediates immunosuppression. PD-1 is amember of the CD28 family of receptors, which includes CD28, CTLA-4,ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1have been identified, Programmed Death Ligand-1 (PD-L1) and ProgrammedDeath Ligand-2 (PD-L2), that are expressed on antigen-presenting cellsas well as many human cancers and have been shown to downregulate T cellactivation and cytokine secretion upon binding to PD-1 (Freeman et al.,2000; Latchman et al., 2001). Unlike CTLA-4, PD-1 primarily functions inperipheral tissues where activated T-cells may encounter theimmunosuppressive PD-L1 (B7-H1) and PD-L2 (B7-DC) ligands expressed bytumor and/or stromal cells (Flies et al., 2011; Topalian et al., 2012a).Inhibition of the PD-1/PD-L1 interaction mediates potent antitumoractivity in preclinical models (U.S. Pat. Nos. 8,008,449 and 7,943,743),and the use of Ab inhibitors of the PD-1/PD-L1 interaction for treatingcancer has entered clinical trials (Brahmer et al., 2010; Flies et al.,2011; Topalian et al., 2012b; Brahmer et al., 2012).

The promise of the emerging field of personalized medicine is thatadvances in pharmacogenomics will increasing be used to tailortherapeutics to defined sub-populations, and ultimately, individualpatients in order to enhance efficacy and minimize adverse effects.Recent successes include, for example, the development of imatinibmesylate (GLEEVEC®), a protein tyrosine kinase inhibitor that inhibitsthe bcr-abl tyrosine kinase, to treat Philadelphia chromosome-positivechronic myelogenous leukemia (CML); crizotinib (XALKORI®) to treat the5% of patients with late-stage non-small cell lung cancers who express amutant anaplastic lymphoma kinase (ALK) gene; and vemurafenib(ZELBORAF®), an inhibitor of mutated B-RAF protein (V600E-BRAF) which isexpressed in around half of melanoma tumors. However, unlike theclinical development of small molecule agents that target discreteactivating mutations found in select cancer populations, a particularchallenge in cancer immunotherapy has been the identification ofmechanism-based predictive biomarkers to enable patient selection andguide on-treatment management. Advances in validating PD-L1 expressionas a biomarker for screening patients for anti-PD-1 immunotherapy aredescribed herein.

SUMMARY OF THE INVENTION

The present disclosure provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises administering to thesubject a composition comprising a therapeutically effective amount ofan agent that reduces or suppresses signaling from an inhibitoryimmunoregulator. In preferred embodiments, the agent is an Ab. In otherpreferred embodiments, the inhibitory immunoregulator is a component ofthe PD-1/PD-L1 signaling pathway. In further preferred embodiments, theAb disrupts the interaction between PD-1 and PD-L1. In certainembodiments, the Ab is an anti-PD-1 Ab of the invention or an anti-PD-L1Ab of the invention. In preferred embodiments, the anti-PD-1 Ab of theinvention is nivolumab (BMS-936558) and the anti-PD-L1 Ab of theinvention is BMS-936559. In certain embodiments, the subject has beenpre-treated for the cancer. In other embodiments, the cancer is anadvanced, metastatic and/or refractory cancer. In preferred embodiments,the administration of the antibody or antigen-binding portion to thesubject thereof induces a durable clinical response in the subject.

This disclosure also provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises: (a) selecting a subjectthat is a suitable candidate for immunotherapy, the selecting comprising(i) optionally providing a test tissue sample obtained from a patientwith cancer of the tissue, the test tissue sample comprising tumor cellsand tumor-infiltrating inflammatory cells, (ii) assessing the proportionof cells in the test tissue sample that express PD-L1 on the cellsurface, and (iii) selecting the subject as a suitable candidate basedon an assessment that the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface exceeds a predetermined thresholdlevel; and (b) administering a composition comprising a therapeuticallyeffective amount of an anti-PD-1 Ab to the selected subject.

The disclosure further provides a method for treatment of a subjectafflicted with cancer, which method comprises: (a) selecting a subjectthat is not suitable for anti-PD-1 Ab immunotherapy, the selectingcomprising (i) optionally providing a test tissue sample obtained from apatient with cancer of the tissue, the test tissue sample comprisingtumor cells and tumor-infiltrating inflammatory cells; (ii) assessingthe proportion of cells in the test tissue sample that express PD-L1 onthe cell surface; and (iii) selecting the subject as not suitable foranti-PD-1 Ab immunotherapy based on an assessment that the proportion ofcells in the test tissue sample that express PD-L1 on the cell surfaceis less than a predetermined threshold level; and (b) administering astandard-of-care therapeutic other than an anti-PD-1 Ab to the selectedsubject.

In addition, the disclosure provides a method for selecting a cancerpatient for immunotherapy with an anti-PD-1 Ab, which method comprises:(a) optionally providing a test tissue sample obtained from a patientwith cancer of the tissue, the test tissue sample comprising tumor cellsand tumor-infiltrating inflammatory cells; (b) assaying the test tissuesample to determine the proportion of cells therein that express PD-L1on the cell surface; (c) comparing the proportion of cells that expressPD-L1 on the cell surface with a predetermined threshold proportion; and(d) selecting the patient for immunotherapy based on an assessment thatPD-L1 is expressed in cells of the test tissue sample.

This disclosure further provides a method for predicting the therapeuticeffectiveness of an anti-PD-1 Ab for treating a cancer patient, whichmethod comprises: (a) optionally providing a test tissue sample obtainedfrom a patient with cancer of the tissue, the test tissue samplecomprising tumor cells and tumor-infiltrating inflammatory cells; (b)assaying the test tissue sample to determine the proportion of cellstherein that express PD-L1 on the cell surface; (c) comparing theproportion of cells that express PD-L1 on the cell surface with apredetermined threshold value; and (d) predicting the therapeuticeffectiveness of the anti-PD-1 Ab, wherein if the proportion of cellsthat express PD-L1 on the cell surface exceeds the threshold proportionthe Ab is predicted to be effective in treating the patient, and whereinif the proportion of cells that express PD-L1 on the cell surface isbelow the threshold proportion the Ab is predicted to not be effectivein treating the patient.

The present disclosure also provides a method for determining animmunotherapeutic regimen comprising an anti-PD-1 Ab for treating acancer patient, which method comprises: (a) optionally providing a testtissue sample obtained from a patient with cancer of the tissue, thetest tissue sample comprising tumor cells and tumor-infiltratinginflammatory cells; (b) assaying the test tissue sample to determine theproportion of cells therein that express PD-L1 on the cell surface; (c)comparing the proportion of cells that express PD-L1 on the cell surfacewith a predetermined threshold proportion; and (d) determining animmunotherapeutic regimen comprising an anti-PD-1 Ab based on thedetermination that the proportion of cells that express PD-L1 on thecell surface exceeds the predetermined threshold proportion.

In certain embodiments of the methods described herein, the test tissuesample is a formalin-fixed and paraffin-embedded (FFPE) sample. Incertain other embodiments, assessing the proportion of cells in the testtissue sample that express PD-L1 on the cell surface is achieved byimmunohistochemical (IHC) staining of the FFPE sample. In preferredembodiments, the mAb 28-8 is used in an automated IHC assay to bind toPD-L1 on the surface of cells in the test tissue sample. In preferredembodiments of any of the methods disclosed herein, the cancer ismelanoma (MEL), renal cell carcinoma (RCC), squamous non-small cell lungcancer (NSCLC), non-squamous NSCLC, colorectal cancer (CRC),castration-resistant prostate cancer (CRPC), hepatocellular carcinoma(HCC), squamous cell carcinoma of the head and neck, carcinomas of theesophagus, ovary, gastrointestinal tract and breast, or a hematologicmalignancy such as multiple myeloma, B-cell lymphoma, T-cell lymphoma,Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, and chronicmyelogenous leukemia.

This invention additionally provides a mAb or antigen-binding portionthereof that binds specifically to a cell surface-expressed human PD-L1antigen in a FFPE tissue sample. In preferred embodiments, the mAb orantigen-binding portion thereof does not bind to a cytoplasmic PD-L1antigen in the FFPE tissue sample. In other preferred embodiments, themonoclonal Ab (mAb) is the rabbit mAb designated 28-8, 28-1, 28-12, 29-8or 20-12.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples which should not beconstrued as limiting. The contents of all cited references, includingscientific articles, newspaper reports, GENBANK® entries, patents andpatent applications cited throughout this application are expresslyincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Cross-competition between 5C4 and other HuMab anti-PD-1mAbs for binding to hPD-1 expressed on CHO cells. A, the 5C4 Fabfragment substantially blocked the binding of mAbs 5C4 itself, as wellas the binding of 2D3 and 7D3; B, the 5C4 Fab fragment substantiallyblocked the binding of mAb 4H1; C, the 5C4 mAb substantially blocked thebinding of mAb 17D8.

FIGS. 2A-2F. Cross-competition of FITC-conjugated human anti-hPD-L1 mAbsfor binding to hPD-L1 expressed on CHO cells. A, Binding of labeled10H10 was partially blocked by 10A5, 11E6 and 13G4 and was significantlyblocked by itself; B, Binding of labeled 3G10 was significantly blockedby each of the tested anti-PD-L1 Abs except 10H10; C, Binding of labeled10A5 was significantly blocked by each of the tested anti-PD-L1 Absexcept 10H10; D, Binding of labeled 11E6 was significantly blocked byeach of the tested anti-PD-L1 Abs except 10H10; and E, Binding oflabeled 12A4 was significantly blocked by each of the tested anti-PD-L1Abs except 10H10; and F, Binding of labeled 13G4 was significantlyblocked by each of the tested anti-PD-L1 Abs except 10H10.

FIG. 3. Cross-competitive inhibition of binding of biotinylated mAb 12A4to ES-2 cells by human anti-hPD-L1 mAbs. Fluorescence of boundbiotin-12A4 is plotted against the concentration of unlabeled hPD-L1HuMabs.

FIG. 4. Spider plot showing activity of anti-PD-1 mAb in patients withtreatment-refractory melanoma (MEL). A representative plot of changes intumor burden over time demonstrates the time course of change in the sumof the longest diameters of target lesions, compared with baseline, in27 MEL patients treated with 5C4 at a dose of 1.0 mg/kg. In the majorityof patients who achieved an objective response (OR), responses weredurable and evident by the end of cycle 3 (6 months) of treatment(vertical dashed line). Tumor regressions followed conventional as wellas “immune-related” patterns of response, such as prolonged reduction intumor burden in the presence of new lesions.

FIG. 5. Activity of anti-PD-1 mAb in patient with metastatic RCC.Partial regression of metastatic RCC in a 57-year-old patient treatedwith 5C4 at 1 mg/kg is illustrated. This patient had previouslyundergone radical surgery and had developed progressive disease afterreceiving sunitinib, temsirolimus, sorafenib, and pazopanib. Arrows showregression of recurrent tumor in the operative field.

FIG. 6. Activity of anti-PD-1 mAb in patient with metastatic MEL. Acomplete response of metastatic MEL is illustrated in a 62-year-oldpatient treated with 5C4 at 3 mg/kg, associated with vitiligo. (i)Pretreatment CT scan, inguinal lymph node metastasis (arrow); (ii) after13 months of treatment. Numerous metastases in the subcutaneous tissueand retroperitoneum also regressed completely (not shown). Vitiligodeveloped after 6 months of treatment; photos taken at 9 months undervisible light (iii) and ultraviolet light (iv). Skin biopsies withimmunohistochemistry for microphthalmia-associated transcription factor(MITF) show melanocytes (arrows) at the epidermal-dermal junction innormal skin (v), and scarce (vi) or absent (vii) melanocytes in skinpartially or fully affected by vitiligo.

FIG. 7. Activity of anti-PD-1 mAb in patient with metastatic NSCLC. Apartial response is illustrated in a patient with metastatic NSCLC(nonsquamous histology) treated with 5C4 at 10 mg/kg. Arrows showinitial progression in pulmonary lesions followed by regression(“immune-related” pattern of response).

FIGS. 8A and 8B. Correlation between tumor PD-L1 expression andanti-PD-1 clinical response. Pretreatment tumor cell surface expressionof PD-L1, as determined by IHC on formalin-fixed paraffin-embeddedspecimens, correlates with OR to PD-1 blockade. Forty-two subjects withadvanced cancers including melanoma, non-small cell lung cancer,colorectal cancer, renal cell cancer, and castration-resistant prostatecancer (n=18, 10, 7, 5, and 2, respectively) were studied. A, There wasa significant correlation of tumor cell surface PD-L1 expression withobjective clinical response. No patients with PD-L1 negative tumorsexperienced an OR. B, Examples of IHC analysis with the anti-PD-L1 mAb5H1 are shown in a melanoma lymph node metastasis (top), a renal cellcancer nephrectomy specimen (middle), and a lung adenocarcinoma brainmetastasis (bottom). All 400× original magnification. Arrows indicateone of many tumor cells in each specimen with surface membrane stainingfor PD-L1. Asterisk indicates a normal glomerulus in the nephrectomyspecimen, which is negative for PD-L1 staining.

FIG. 9. Graphical comparison of the binding of mAbs 28-8 and 5H1 toPD-L1 antigen in tumor tissues by histoscore analysis. The rabbit mAb28-8 showed higher histoscores in 7 out of 10 samples tested.

FIGS. 10A-10D. Spider plot showing activity of anti-PD-L1 mAb inpatients with treatment-refractory MEL and NSCLC. Representative plotsdemonstrate the time course of target lesion tumor burden over time inpatients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10mg/kg (C) and in patients with NSCLC treated at 10 mg/kg (D). In themajority of patients who achieved ORs, responses were durable and wereevident by the end of cycle 2 (3 months) of treatment, irrespective ofdose or tumor type. Tumor regressions followed conventional as well as“immune-related” patterns of response.

FIG. 11. Complete response in a patient with melanoma treated withBMS-936559 at 3 mg/kg. Circles indicate an initial increase in the sizeof pulmonary nodules at 6 weeks and 3 months followed by completeregression at 10 months (“immune-related” pattern of response).

FIG. 12. Complete response in a patient with melanoma treated withBMS-936559 at 1 mg/kg. This patient developed an isolated brainmetastasis 3 months after initiation of treatment that was successfullytreated with stereotactic radiosurgery. A partial response in abdominaldisease (circled) was noted at 8 months, with no evidence of disease at15 months.

FIG. 13. Partial response in a patient with NSCLC (non-squamoushistology) treated with BMS-936559 at 10 mg/kg. Note the response indisease in right lung pleura and liver.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for immunotherapy of a subjectafflicted with diseases such as cancer or an infectious disease, whichmethods comprise administering to the subject a composition comprising atherapeutically effective amount of a compound or agent that potentiatesan endogenous immune response, either stimulating the activation of theendogenous response or inhibiting the suppression of the endogenousresponse. More specifically, the disclosure provides methods forpotentiating an endogenous immune response in a subject afflicted withcancer so as to thereby treat the patient, which method comprisesadministering to the subject a therapeutically effective amount of an Abor an antigen-binding portion thereof that inhibits signaling from aninhibitory immunoregulator. In certain embodiments, the inhibitoryimmunoregulator is a component of the PD-1/PD-L1 signaling pathway.Accordingly, certain embodiments of the invention provide methods forimmunotherapy of a subject afflicted with cancer, which methods compriseadministering to the subject a therapeutically effective amount of an Abor an antigen-binding portion thereof that disrupts the interactionbetween the PD-1 receptor and its ligand, PD-L1. In certain preferredembodiments, the Ab or antigen-binding portion thereof bindsspecifically to PD-1. In other preferred embodiments, the Ab orantigen-binding portion thereof binds specifically to PD-L1. In certainother embodiments, the subject is selected as suitable for immunotherapyin a method comprising measuring the surface expression of PD-L1 in atest tissue sample obtained from a patient with cancer of the tissue,for example, determining the proportion of cells in the test tissuesample that express PD-L1 on the cell surface, and selecting the patientfor immunotherapy based on an assessment that PD-L1 is expressed on thesurface of cells in the test tissue sample.

Terms

In order that the present disclosure may be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

“Administering” refers to the physical introduction of a compositioncomprising a therapeutic agent to a subject, using any of the variousmethods and delivery systems known to those skilled in the art.Preferred routes of administration for Abs of the invention includeintravenous, intramuscular, subcutaneous, intraperitoneal, spinal orother parenteral routes of administration, for example by injection orinfusion. The phrase “parenteral administration” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intralymphatic,intralesional, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an Ab of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended or undesirable sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. For example, an adverse event may be associated withactivation of the immune system or expansion of immune system cells(e.g., T cells) in response to a treatment. A medical treatment may haveone or more associated AEs and each AE may have the same or differentlevel of severity. Reference to methods capable of “altering adverseevents” means a treatment regime that decreases the incidence and/orseverity of one or more AEs associated with the use of a differenttreatment regime.

An “antibody” (Ab) shall include, without limitation, a glycoproteinimmunoglobulin which binds specifically to an antigen and comprises atleast two heavy (H) chains and two light (L) chains interconnected bydisulfide bonds, or an antigen-binding portion thereof. Each H chaincomprises a heavy chain variable region (abbreviated herein as V_(H))and a heavy chain constant region. The heavy chain constant regioncomprises three constant domains, C_(H1), C_(H2) and C_(H3). Each lightchain comprises a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprises one constant domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the Abs may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁵ to10⁻¹¹ M⁻¹ or less. Any K_(D) greater than about 10⁻⁴ M⁻¹ is generallyconsidered to indicate nonspecific binding. As used herein, an Ab that“binds specifically” to an antigen refers to an Ab that binds to theantigen and substantially identical antigens with high affinity, whichmeans having a K_(D) of 10⁻⁷ M or less, preferably 10⁻⁸ M or less, evenmore preferably 5×10⁻⁹ M or less, and most preferably between 10⁻⁸ M and10⁻¹⁰ M or less, but does not bind with high affinity to unrelatedantigens. An antigen is “substantially identical” to a given antigen ifit exhibits a high degree of sequence identity to the given antigen, forexample, if it exhibits at least 80%, at least 90%, preferably at least95%, more preferably at least 97%, or even more preferably at least 99%sequence identity to the sequence of the given antigen. By way ofexample, an Ab that binds specifically to human PD-1 may also havecross-reactivity with PD-1 antigens from certain primate species but maynot cross-react with PD-1 antigens from certain rodent species or withan antigen other than PD-1, e.g., a human PD-L1 antigen.

An immunoglobulin may derive from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. IgGsubclasses are also well known to those in the art and include but arenot limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to theAb class or subclass (e.g., IgM or IgG1) that is encoded by the heavychain constant region genes. The term “antibody” includes, by way ofexample, both naturally occurring and non-naturally occurring Abs;monoclonal and polyclonal Abs; chimeric and humanized Abs; human ornonhuman Abs; wholly synthetic Abs; and single chain Abs. A nonhuman Abmay be humanized by recombinant methods to reduce its immunogenicity inman. Where not expressly stated, and unless the context indicatesotherwise, the term “antibody” also includes an antigen-binding fragmentor an antigen-binding portion of any of the aforementionedimmunoglobulins, and includes a monovalent and a divalent fragment orportion, and a single chain Ab.

An “isolated antibody” refers to an Ab that is substantially free ofother Abs having different antigenic specificities (e.g., an isolated Abthat binds specifically to PD-1 is substantially free of Abs that bindspecifically to antigens other than PD-1). An isolated Ab that bindsspecifically to PD-1 may, however, have cross-reactivity to otherantigens, such as PD-1 molecules from different species. Moreover, anisolated Ab may be substantially free of other cellular material and/orchemicals. By comparison, an “isolated” nucleic acid refers to a nucleicacid composition of matter that is markedly different, i.e., has adistinctive chemical identity, nature and utility, from nucleic acids asthey exist in nature. For example, an isolated DNA, unlike native DNA,is a free-standing portion of a native DNA and not an integral part of alarger structural complex, the chromosome, found in nature. Further, anisolated DNA, unlike native genomic DNA, can typically be used inapplications or methods for which native genomic DNA is unsuited, e.g.,as a PCR primer or a hybridization probe for, among other things,measuring gene expression and detecting biomarker genes or mutations fordiagnosing disease or assessing the efficacy of a therapeutic. Anisolated nucleic acid may be purified so as to be substantially free ofother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, using standard techniques well known in theart. Examples of isolated nucleic acids include fragments of genomicDNA, PCR-amplified DNA, cDNA and RNA.

The term “monoclonal antibody” (“mAb”) refers to a preparation of Abmolecules of single molecular composition, i.e., Ab molecules whoseprimary sequences are essentially identical, and which exhibits a singlebinding specificity and affinity for a particular epitope. A mAb is anexample of an isolated Ab. MAbs may be produced by hybridoma,recombinant, transgenic or other techniques known to those skilled inthe art.

A “human” antibody (HuMAb) refers to an Ab having variable regions inwhich both the framework and CDR regions are derived from human germlineimmunoglobulin sequences. Furthermore, if the Ab contains a constantregion, the constant region also is derived from human germlineimmunoglobulin sequences. The human Abs of the invention may includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody,” as used herein, is not intended to include Abs inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. The terms “human” Abs and “fully human” Abs and are usedsynonymously.

A “humanized” antibody refers to an Ab in which some, most or all of theamino acids outside the CDR domains of a non-human Ab are replaced withcorresponding amino acids derived from human immunoglobulins. In oneembodiment of a humanized form of an Ab, some, most or all of the aminoacids outside the CDR domains have been replaced with amino acids fromhuman immunoglobulins, whereas some, most or all amino acids within oneor more CDR regions are unchanged. Small additions, deletions,insertions, substitutions or modifications of amino acids arepermissible as long as they do not abrogate the ability of the Ab tobind to a particular antigen. A “humanized” Ab retains an antigenicspecificity similar to that of the original Ab.

A “chimeric antibody” refers to an Ab in which the variable regions arederived from one species and the constant regions are derived fromanother species, such as an Ab in which the variable regions are derivedfrom a mouse Ab and the constant regions are derived from a human Ab.

An “antigen-binding portion” of an Ab (also called an “antigen-bindingfragment”) refers to one or more fragments of an Ab that retain theability to bind specifically to the antigen bound by the whole Ab.

A “cancer” refers a broad group of various diseases characterized by theuncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth divide and grow results in the formation ofmalignant tumors that invade neighboring tissues and may alsometastasize to distant parts of the body through the lymphatic system orbloodstream.

An “immune response” refers to the action of a cell of the immune system(for example, T lymphocytes, B lymphocytes, natural killer (NK) cells,macrophages, eosinophils, mast cells, dendritic cells and neutrophils)and soluble macromolecules produced by any of these cells or the liver(including Abs, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom a vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues.

An “immunoregulator” refers to a substance, an agent, a signalingpathway or a component thereof that regulates an immune response.“Regulating,” “modifying” or “modulating” an immune response refers toany alteration in a cell of the immune system or in the activity of suchcell. Such regulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunoregulators have been identified,some of which may have enhanced function in the cancer microenvironment.

The term “immunotherapy” refers to the treatment of a subject afflictedwith, or at risk of contracting or suffering a recurrence of, a diseaseby a method comprising inducing, enhancing, suppressing or otherwisemodifying an immune response. “Treatment” or “therapy” of a subjectrefers to any type of intervention or process performed on, or theadministration of an active agent to, the subject with the objective ofreversing, alleviating, ameliorating, inhibiting, slowing down orpreventing the onset, progression, development, severity or recurrenceof a symptom, complication, condition or biochemical indicia associatedwith a disease.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency may be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

A “predetermined threshold value,” relating to cell surface PD-L1expression, refers to the proportion of cells in a test tissue samplecomprising tumor cells and tumor-infiltrating inflammatory cells abovewhich the sample is scored as being positive for cell surface PD-L1expression. For cell surface expression assayed by IHC with the mAb28-8, the predetermined threshold value for cells expressing PD-L1 onthe cell surface ranges from at least about 0.01% to at least about 20%of the total number of cells. In preferred embodiments, thepredetermined threshold value for cells expressing PD-L1 on the cellsurface ranges from at least about 0.1% to at least about 10% of thetotal number of cells. More preferably, the predetermined thresholdvalue is at least about 5%. Even more preferably, the predeterminedthreshold value is at least about 1%.

The “Programmed Death-1 (PD-1)” receptor refers to an immunoinhibitoryreceptor belonging to the CD28 family. PD-1 is expressed predominantlyon previously activated T cells in vivo, and binds to two ligands, PD-L1and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1),variants, isoforms, and species homologs of hPD-1, and analogs having atleast one common epitope with hPD-1. The complete hPD-1 sequence can befound under GENBANK® Accession No. U64863.

“Programmed Death Ligand-1 (PD-L1)” is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that downregulateT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GENBANK® Accession No. Q9NZQ7.

A “signal transduction pathway” or “signaling pathway” refers to thebiochemical relationship between a variety of signal transductionmolecules that play a role in the transmission of a signal from oneportion of a cell to another portion of the cell. A “cell surfacereceptor” includes, for example, molecules and complexes of moleculesthat are located on the surface of a cell and are capable of receiving asignal and transmitting such a signal across the plasma membrane of acell. An example of a cell surface receptor of the present invention isthe PD-1 receptor, which is located on the surface of activated B cells,activated T cells and myeloid cells, and transmits a signal that resultsin a decrease in tumor-infiltrating lymphocytes and a decrease in T cellproliferation. An “inhibitor” of signaling refers to a compound or agentthat antagonizes or reduces the initiation, reception or transmission ofa signal, be that signal stimulatory or inhibitory, by any component ofa signaling pathway such as a receptor or its ligand.

A “subject” includes any human or nonhuman animal. The term “nonhumananimal” includes, but is not limited to, vertebrates such as nonhumanprimates, sheep, dogs, cats, rabbits, ferrets, rodents such as mice,rats and guinea pigs, avian species such as chickens, amphibians, andreptiles. In preferred embodiments, the subject is a mammal such as anonhuman primate, sheep, dog, cat, rabbit, ferret or rodent. In morepreferred embodiments, the subject is a human. The terms, “subject,”“patient” and “individual” are used interchangeably herein.

A “therapeutically effective amount” or “therapeutically effectivedosage” of a drug or therapeutic agent, such as an Ab of the invention,is any amount of the drug that, when used alone or in combination withanother therapeutic agent, protects a subject against the onset of adisease or promotes disease regression evidenced by a decrease inseverity of disease symptoms, an increase in frequency and duration ofdisease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction. The ability of a therapeuticagent to promote disease regression can be evaluated using a variety ofmethods known to the skilled practitioner, such as in human subjectsduring clinical trials, in animal model systems predictive of efficacyin humans, or by assaying the activity of the agent in in vitro assays.

By way of example, an anti-cancer agent promotes cancer regression in asubject. In preferred embodiments, a therapeutically effective amount ofthe drug promotes cancer regression to the point of eliminating thecancer. “Promoting cancer regression” means that administering aneffective amount of the drug, alone or in combination with ananti-neoplastic agent, results in a reduction in tumor growth or size,necrosis of the tumor, a decrease in severity of at least one diseasesymptom, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. In addition, the terms “effective” and “effectiveness” withregard to a treatment includes both pharmacological effectiveness andphysiological safety. Pharmacological effectiveness refers to theability of the drug to promote cancer regression in the patient.Physiological safety refers to the level of toxicity, or other adversephysiological effects at the cellular, organ and/or organism level(adverse effects) resulting from administration of the drug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount of the drug preferably inhibits cell growth or tumorgrowth by at least about 20%, more preferably by at least about 40%,even more preferably by at least about 60%, and still more preferably byat least about 80% relative to untreated subjects. In other preferredembodiments of the invention, tumor regression may be observed andcontinue for a period of at least about 20 days, more preferably atleast about 40 days, or even more preferably at least about 60 days.Notwithstanding these ultimate measurements of therapeuticeffectiveness, evaluation of immunotherapeutic drugs must also makeallowance for “immune-related” response patterns.

An “immune-related” response pattern refers to a clinical responsepattern often observed in cancer patients treated with immunotherapeuticagents that produce antitumor effects by inducing cancer-specific immuneresponses or by modifying native immune processes. This response patternis characterized by a beneficial therapeutic effect that follows aninitial increase in tumor burden or the appearance of new lesions, whichin the evaluation of traditional chemotherapeutic agents would beclassified as disease progression and would be synonymous with drugfailure. Accordingly, proper evaluation of immunotherapeutic agents mayrequire long-term monitoring of the effects of these agents on thetarget disease.

A therapeutically effective amount of a drug includes a“prophylactically effective amount,” which is any amount of the drugthat, when administered alone or in combination with an anti-neoplasticagent to a subject at risk of developing a cancer (e.g., a subjecthaving a pre-malignant condition) or of suffering a recurrence ofcancer, inhibits the development or recurrence of the cancer. Inpreferred embodiments, the prophylactically effective amount preventsthe development or recurrence of the cancer entirely. “Inhibiting” thedevelopment or recurrence of a cancer means either lessening thelikelihood of the cancer's development or recurrence, or preventing thedevelopment or recurrence of the cancer entirely.

A “tumor-infiltrating inflammatory cell” is any type of cell thattypically participates in an inflammatory response in a subject andwhich infiltrates tumor tissue. Such cells include tumor-infiltratinglymphocytes (TILs), macrophages, monocytes, eosinophils, histiocytes anddendritic cells.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 20%. Furthermore, particularly with respect to biologicalsystems or processes, the terms can mean up to an order of magnitude orup to 5-fold of a value. When particular values or compositions areprovided in the application and claims, unless otherwise stated, themeaning of “about” or “comprising essentially of” should be assumed tobe within an acceptable error range for that particular value orcomposition.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

Various aspects of the invention are described in further detail in thefollowing subsections.

Antibodies of the Invention

Abs of the present invention include a variety of Abs having structuraland functional properties described herein, including high-affinitybinding to PD-1 or PD-L1, respectively. These Abs may be used, forexample, as therapeutic Abs to treat subjects afflicted with disease oras reagents in diagnostic assays to detect their cognate antigens. HumanmAbs (HuMAbs) that bind specifically to PD-1 (e.g., bind to human PD-1and may cross-react with PD-1 from other species, such as cynomolgusmonkey) with high affinity have been disclosed in U.S. Pat. No.8,008,449, and HuMAbs that bind specifically to PD-L1 with high affinityhave been disclosed in U.S. Pat. No. 7,943,743. The Abs of the inventioninclude, but are not limited to, all of the anti-PD-1 and anti-PD-L1 Absdisclosed in U.S. Pat. Nos. 8,008,449 and 7,943,743, respectively. Otheranti-PD-1 mAbs have been described in, for example, U.S. Pat. Nos.7,488,802 and 8,168,757, and anti-PD-L1 mAbs have been described in, forexample, U.S. Pat. Nos. 7,635,757 and 8,217,149, and U.S. PublicationNo. 2009/0317368. To the extent these anti-PD-1 and anti-PD-L1 mAbsexhibit the structural and functional properties disclosed herein forantibodies of the invention, they too are included as antibodies of theinvention.

Anti-PD-1 Antibodies of the Invention

Each of the anti-PD-1 HuMAbs disclosed in U.S. Pat. No. 8,008,449 hasbeen demonstrated to exhibit one or more of the followingcharacteristics: (a) binds to human PD-1 with a K_(D) of 1×10⁻⁷ M orless, as determined by surface plasmon resonance using a BIACORE®biosensor system; (b) does not substantially bind to human CD28, CTLA-4or ICOS; (c) increases T-cell proliferation in a Mixed LymphocyteReaction (MLR) assay; (d) increases interferon-γ production in an MLRassay; (e) increases IL-2 secretion in an MLR assay; (f) binds to humanPD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1and/or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses;(i) stimulates Ab responses; and (j) inhibits tumor cell growth in vivo.Anti-PD-1 Abs of the present invention include mAbs that bindspecifically to human PD-1 and exhibit at least one, preferably at leastfive, of the preceding characteristics. U.S. Pat. No. 8,008,449exemplifies seven anti-PD-1 HuMAbs: 17D8, 2D3, 4H1, 5C4 (also referredto herein as nivolumab or BMS-936558), 4A11, 7D3 and 5F4. Isolated DNAmolecules encoding the heavy and light chain variable regions of theseAbs have been sequenced, from which the amino acid sequences of thevariable regions were deduced. The V_(H) amino acid sequences of 17D8,2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are provided herein as SEQ ID NOs. 1,2, 3, 4, 5, 6 and 7, respectively. The V_(L) amino acid sequences of17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are provided herein as SEQ IDNOs. 8, 9, 10, 11, 12, 13 and 14, respectively.

Preferred anti-PD-1 Abs of the present invention include the anti-PD-1HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. These preferred Abs bindspecifically to human PD-1 and comprise: (a) a human heavy chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 1 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 8; (b) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 2 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 9; (c) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 3 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 10; (d) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 4 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 11; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 5 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 12; (f) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 6 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 13; or (g) a humanheavy chain variable region comprising consecutively linked amino acidshaving the sequence set forth in SEQ ID NO: 7 and a human light chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 14.

Given that each of these Abs can bind to PD-1, the V_(H) and V_(L)sequences can be “mixed and matched” to create other anti-PD-1 Abs ofthe invention. PD-1 binding of such “mixed and matched” Abs can betested using binding assays, e.g., enzyme-linked immunosorbent assays(ELISAs), western blots, radioimmunoassays and BIACORE® analysis thatare well known in the art (see, e.g., U.S. Pat. No. 8,008,449).Preferably, when V_(H) and V_(L) chains are mixed and matched, a V_(H)sequence from a particular V_(H)N_(L) pairing is replaced with astructurally similar V_(H) sequence. Likewise, preferably a V_(L)sequence from a particular V_(H)N_(L) pairing is replaced with astructurally similar V_(L) sequence. Accordingly, anti-PD-1 Abs of theinvention include an isolated mAb or antigen-binding portion thereofcomprising: (a) a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs. 1, 2, 3, 4,5, 6 and 7, and (b) a light chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs. 8, 9,10, 11, 12, 13 and 14, wherein the Ab specifically binds PD-1,preferably human PD-1.

The CDR domains of the above Abs have been delineated using the Kabatsystem, and these Abs may also be defined by combinations of their 3heavy chain and 3 light chain CDRs (see U.S. Pat. No. 8,008,449). Sinceeach of these Abs can bind to PD-1 and antigen-binding specificity isprovided primarily by the CDR1, CDR2, and CDR3 regions, the V_(H) CDR1,CDR2, and CDR3 sequences and V_(κ) CDR1, CDR2, and CDR3 sequences can be“mixed and matched” (i.e., CDRs from different Abs can be mixed andmatch, although each Ab must contain a V_(H) CDR1, CDR2, and CDR3 and aV_(κ) CDR1, CDR2, and CDR3) to create other anti-PD-1 Abs that alsoconstitute Abs of the invention. PD-1 binding of such “mixed andmatched” Abs can be tested using the binding assays described above(e.g., ELISAs, western blots, radioimmunoassays and BIACORE® analysis).

Abs of the invention also include isolated Abs that bind specifically toPD-1 and comprise a heavy chain variable region derived from aparticular germline heavy chain immunoglobulin and/or a light chainvariable region derived from a particular germline light chainimmunoglobulin. Specifically, in certain embodiments, Abs of theinvention include isolated Abs comprising: (a) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 3-33 or 4-39 germline sequence, and/or alight chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L6, or L15 germlinesequence. The amino acid sequences of the V_(H) and V_(κ) regionsencoded by the V_(H) 3-33, V_(H) 4-39, V_(κ) L6 and V_(κ) L15 germlinegenes are provided in U.S. Pat. No. 8,008,449.

As used herein, an Ab can be identified as comprising a heavy or a lightchain variable region that is “derived from” a particular human germlineimmunoglobulin by comparing the amino acid sequence of the human Ab tothe amino acid sequences encoded by human germline immunoglobulin genes,and selecting the human germline immunoglobulin sequence that is closestin sequence (i.e., greatest percentage of sequence identity) to thesequence of the human Ab. A human Ab that is “derived from” a particularhuman germline immunoglobulin may contain amino acid differences ascompared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a selected human Ab is generally atleast 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the human Ab as being human when compared to thegermline immunoglobulin amino acid sequences of other species (e.g.,murine germline sequences). In certain cases, a human Ab may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene.

In certain embodiments, the sequence of a human Ab derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In other embodiments, the human Ab maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifferences from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Preferred Abs of the invention also include isolated Abs orantigen-binding portions thereof comprising: (a) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 3-33 germline sequence, and a light chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(κ) L6 germline sequence; or (b) a heavychain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(H) 4-39 germline sequence, anda light chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L15 germlinesequence. Examples of Abs having a V_(H) and a V_(κ) derived from V_(H)3-33 and V_(κ) L6 germline sequences, respectively, include 17D8, 2D3,4H1, 5C4, and 7D3. Examples of Abs having V_(H) and V_(κ) regionsderived from V_(H) 4-39 and V_(κ) L15 germline sequences, respectively,include 4A11 and 5F4.

In yet other embodiments, anti-PD-1 Abs of the invention comprise heavyand light chain variable regions having amino acid sequences that arehighly similar or homologous to the amino acid sequences of thepreferred anti-PD-1 Abs described herein, wherein the Ab retains thefunctional properties of the preferred anti-PD-1 Abs of the invention.For example, Abs of the invention include mAbs comprising a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises consecutively linked amino acids havinga sequence that is at least 80% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs. 1, 2, 3, 4, 5, 6 and7, and the light chain variable region comprises consecutively linkedamino acids having a sequence that is at least 80% identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs. 8, 9,10, 11, 12, 13 and 14. In other embodiments, the V_(H) and/or V_(L)amino acid sequences may exhibit at least 85%, 90%, 95%, 96%, 97%, 98%or 99% identity to the sequences set forth above.

As used herein, the percent sequence identity (also referred to as thepercent sequence homology) between two sequences (amino acid ornucleotide sequences) is a function of the number of identical positionsshared by the sequences relative to the length of the sequences compared(i.e., % identity=number of identical positions/total number ofpositions being compared×100), taking into account the number of anygaps, and the length of each such gap, introduced to maximize the degreeof sequence identity between the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using mathematical algorithms that are well know tothose of ordinary skill in the art (see, e.g., U.S. Pat. No. 8,008,449).

Antibodies having very similar amino acid sequences are likely to haveessentially the same functional properties where the sequencedifferences are conservative modifications. As used herein,“conservative sequence modifications” refer to amino acid modificationsthat do not significantly affect the binding characteristics of the Abcontaining the amino acid sequence. Such conservative modificationsinclude amino acid substitutions, additions and deletions. Conservativeamino acid substitutions are substitutions in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Thus, for example, one or more amino acid residues within the CDRregions of an Ab of the invention can be replaced with other amino acidresidues from the same side chain family and the altered Ab can betested for retained function using functional assays that are well knownin the art. Accordingly, certain embodiments of the anti-PD-1 Abs of theinvention comprise heavy and light chain variable regions eachcomprising CDR1, CDR2 and CDR3 domains, wherein one or more of these CDRdomains comprise consecutively linked amino acids having sequences thatare the same as the CDR sequences of the preferred anti-PD-1 Absdescribed herein (e.g., 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4), orconservative modifications thereof, and wherein the Abs retain thedesired functional properties of the preferred anti-PD-1 Abs of theinvention.

Further, it is well known in the art that the heavy chain CDR3 is theprimary determinant of binding specificity and affinity of an Ab, andthat multiple Abs can predictably be generated having the same bindingcharacteristics based on a common CDR3 sequence (see, e.g., Klimka etal., 2000; Beiboer et al., 2000; Rader et al., 1998; Barbas et al.,1994; Barbas et al., 1995; Ditzel et al., 1996; Berezov et al., 2001;Igarashi et al., 1995; Bourgeois et al., 1998; Levi et al., 1993;Polymenis et al., 1994; and Xu et al., 2000). The foregoing publicationsdemonstrate that, in general, once the heavy chain CDR3 sequence of agiven Ab is defined, variability in the other five CDR sequences willnot greatly affect the binding specificity of that Ab. Thus, Abs of theinvention comprising 6 CDRs can be defined by specifying the sequence ofthe heavy chain CDR3 domain.

Anti-PD-1 Abs of the invention also include isolated Abs that bindspecifically to human PD-1 and cross-compete for binding to human PD-1with any of HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. Thus,anti-PD-1 Abs of the invention include isolated Abs or antigen-bindingportions thereof that cross-compete for binding to PD-1 with a referenceAb or a reference antigen-binding portion thereof comprising: (a) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 1 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 8; (b) a human heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 2 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 9; (c) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 3 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 10; (d) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 4 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 11; (e) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 5 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 12; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 6 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 13; or (g) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 7 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEO ID NO: 14.

The ability of Abs to cross-compete for binding to an antigen indicatesthat these Abs bind to the same epitope region (i.e., the same or anoverlapping epitope) of the antigen and sterically hinder the binding ofother cross-competing Abs to that particular epitope region. Thus, theability of a test Ab to competitively inhibit the binding of, forexample, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, to human PD-1demonstrates that the test Ab binds to the same epitope region of humanPD-1 as 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, respectively. Allisolated Abs that bind to the same epitope region of human PD-1 as doesHuMAb 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 are included among the Absof the invention. These cross-competing Abs are expected to have verysimilar functional properties by virtue of their binding to the sameepitope region of PD-1. For example, cross-competing anti-PD-1 mAbs 5C4,2D3, 7D3, 4H1 and 17D8 have been shown to have similar functionalproperties (see U.S. Pat. No. 8,008,449 at Examples 3-7). The higher thedegree of cross-competition, the more similar will the functionalproperties be. Further, cross-competing Abs can be readily identifiedbased on their ability to cross-compete with 17D8, 2D3, 4H1, 5C4, 4A11,7D3 or 5F4 in standard PD-1 binding assays. For example, BIACORE®analysis, ELISA assays or flow cytometry may be used to demonstratecross-competition with the Abs of the invention (see, e.g., Examples 1and 2). In preferred embodiments, the Abs that cross-compete for bindingto human PD-1 with, or bind to the same epitope region of human PD-1 as,17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 are mAbs, preferably chimeric Abs,or more preferably humanized or human Abs. Such human mAbs can beprepared and isolated as described in U.S. Pat. No. 8,008,449. Dataprovided in Example 1 show that 5C4 or a Fab fragment thereofcross-competes with each of 2D3, 7D3, 4H1 or 17D8 for binding to hPD-1expressed on the surface of a cell, indicating that all five anti-PD-1mAbs bind to the same epitope region of hPD-1 (FIGS. 1A-1C).

An anti-PD-1 Ab of the invention further can be prepared using an Abhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified Ab, which modified Ab mayhave altered properties from the starting Ab. An Ab can be engineered bymodifying one or more residues within one or both variable regions(i.e., V_(H) and/or V_(L)), for example within one or more CDR regionsand/or within one or more framework regions. Additionally oralternatively, an Ab can be engineered by modifying residues within theconstant region(s), for example, to alter the effector function(s) ofthe Ab. Specific modifications to Abs include CDR grafting,site-specific mutation of amino acid residues within the V_(H) and/orV_(κ) CDR1, CDR2 and/or CDR3 regions to thereby improve one or morebinding properties (e.g., affinity) of the Ab, site-specific mutation ofamino acid residues within the V_(H) and/or V_(κ) framework regions todecrease the immunogenicity of the Ab, modifications within the Fcregion, typically to alter one or more functional properties of the Ab,such as serum half-life, complement fixation, Fc receptor binding,and/or antigen-dependent cellular cytotoxicity, and chemicalmodification such as pegylation or alteration in glycosylation patternsto increase or decrease the biological (e.g., serum) half life of theAb. Specific examples of such modifications and methods of engineeringAbs are described in detail in U.S. Pat. No. 8,008,449. Anti-PD-1 Abs ofthe invention include all such engineered Abs that bind specifically tohuman PD-1 and are obtained by modification of any of theabove-described anti-PD-1 Abs.

Anti-PD-1 Abs of the invention also include antigen-binding portions ofthe above Abs. It has been amply demonstrated that the antigen-bindingfunction of an Ab can be performed by fragments of a full-length Ab.Examples of binding fragments encompassed within the term“antigen-binding portion” of an Ab include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; and (iv) a Fvfragment consisting of the V_(L) and V_(H) domains of a single arm of anAb.

These fragments, obtained initially through proteolysis with enzymessuch as papain and pepsin, have been subsequently engineered intomonovalent and multivalent antigen-binding fragments. For example,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker peptide that enables them to be made as a singleprotein chain in which the V_(L) and V_(H) regions pair to formmonovalent molecules known as single chain variable fragments (scFv).Divalent or bivalent scFvs (di-scFvs or bi-scFvs) can be engineered bylinking two scFvs in within a single peptide chain known as a tandemscFv which contains two V_(H) and two V_(L) regions. ScFv dimers andhigher multimers can also be created using linker peptides of fewer than10 amino acids that are too short for the two variable regions to foldtogether, which forces the scFvs to dimerize and produce diabodies orform other multimers. Diabodies have been shown to bind to their cognateantigen with much higher affinity than the corresponding scFvs, havingdissociation constants up to 40-fold lower than the K_(D) values for thescFvs. Very short linkers (≤3 amino acids) lead to the formation oftrivalent triabodies or tetravalent tetrabodies that exhibit even higheraffinities for to their antigens than diabodies. Other variants includeminibodies, which are scFv-C_(H3) dimers, and larger scFv-Fc fragments(scFv-C_(H2)-C_(H3) dimers), and even an isolated CDR may exhibitantigen-binding function. These Ab fragments are engineered usingconventional recombinant techniques known to those of skill in the art,and the fragments are screened for utility in the same manner as areintact Abs. All of the above proteolytic and engineered fragments of Absand related variants (see Hollinger et al., 2005; Olafsen et al., 2010,for further details) are intended to be encompassed within the term“antigen-binding portion” of an Ab.

Anti-PD-L1 Antibodies of the Invention

Each of the anti-PD-L1 HuMAbs disclosed in U.S. Pat. No. 7,943,743 hasbeen demonstrated to exhibit one or more of the followingcharacteristics (a) binds to human PD-L1 with a K_(D) of 1×10⁻⁷ M orless; (b) increases T-cell proliferation in a Mixed Lymphocyte Reaction(MLR) assay; (c) increase interferon-γ production in an MLR assay; (d)increase IL-2 secretion in an MLR assay; (e) stimulates Ab responses;(f) inhibits the binding of PD-L1 to PD-1; and (g) reverses thesuppressive effect of T regulatory cells on T cell effector cells and/ordendritic cells. Anti-PD-L1 Abs of the present invention include mAbsthat bind specifically to human PD-L1 and exhibit at least one,preferably at least four, of the preceding characteristics.

U.S. Pat. No. 7,943,743 exemplifies ten anti-PD-1 HuMAbs: 3G10, 12A4(also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1,11E6, 12B7, and 13G4. Isolated DNA molecules encoding the heavy andlight chain variable regions of these Abs have been sequenced, fromwhich the amino acid sequences of the variable regions were deduced. TheV_(H) amino acid sequences of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1,11E6, 12B7, and 13G4 are shown in SEQ ID NOs. 15, 16, 17, 18, 19, 20,21, 22, 23 and 24, respectively, whereas their V_(L) amino acidsequences are shown in SEQ ID NOs. 25, 26, 27, 28, 29, 30, 31, 32, 33and 34, respectively.

Preferred anti-PD-L1 Abs of the present invention include the anti-PD-L1HuMAbs 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4.These preferred Abs bind specifically to human PD-L1 and comprise: (a) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 15 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 25; (b) a human heavy chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 16 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 26; (c) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 17 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 27; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 18 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 28; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 19 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 29; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 20 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 30; (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 21 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 31; (h) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 22 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 32; (i) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 23 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEO ID NO: 33; or (j) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 24 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 34.

Given that each of these Abs can bind to PD-L1, the V_(H) and V_(L)sequences can be “mixed and matched” to create other anti-PD-L1 Abs ofthe invention. PD-L1 binding of such “mixed and matched” Abs can betested using binding assays e.g., ELISAs, western blots,radioimmunoassays and BIACORE® analysis that are well known in the art(see, e.g., U.S. Pat. No. 7,943,743). Preferably, when V_(H) and V_(L)chains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence. Accordingly, Abs of the invention also include a mAb, orantigen binding portion thereof, comprising a heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in any of SEQ ID NOs. 15, 16, 17, 18, 19, 20, 21, 22, 23 or24, and a light chain variable region comprising consecutively linkedamino acids having the sequence set forth in any of SEQ ID NOs. 25, 26,27, 28, 29, 30, 31, 32, 33 or 34, wherein the Ab binds specifically toPD-L1, preferably human PD-L1.

The CDR domains of the above anti-PD-L1 HuMAbs have been delineatedusing the Kabat system, and these Abs may also be defined bycombinations of their 3 heavy chain and 3 light chain CDRs (see U.S.Pat. No. 7,943,743). Since each of these Abs can bind to PD-L1 andantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(κ) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent Abs can be mixed and match, although each Ab must contain aV_(H) CDR1, CDR2, and CDR3 and a V_(κ) CDR1, CDR2, and CDR3) to createother anti-PD-1 Abs that also constitute Abs of the invention. PD-L1binding of such “mixed and matched” Abs can be tested using, forexample, ELISAs, western blots, radioimmunoassays and BIACORE® analysis.

Antibodies of the invention also include Abs that bind specifically toPD-L1 and comprise a heavy chain variable region derived from aparticular germline heavy chain immunoglobulin and/or a light chainvariable region derived from a particular germline light chainimmunoglobulin. Specifically, in certain embodiments, Abs of theinvention include Abs comprising: (a) a heavy chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(H) 1-18, 1-69, 1-3 or 3-9 germline sequence, and/or alight chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L6, L15, A27 or L18germline sequence. The amino acid sequences of the V_(H) and V_(κ)regions encoded by the V_(H) 1-18, V_(H) 1-3, V_(H) 1-69, V_(H) 3-9,V_(κ) L6, V_(κ) L15 and V_(κ) A27 germline genes are provided in U.S.Pat. No. 7,943,743.

Preferred Abs of the invention include isolated Abs or antigen-bindingportions thereof comprising: (a) a heavy chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(H) 1-18 germline sequence, and a light chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(κ) L6 germline sequence; (b) a heavy chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(H) 1-69 germline sequence, and a lightchain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(κ) L6 germline sequence; (c) aheavy chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(H) 1-3 germlinesequence, and a light chain variable region that comprises consecutivelylinked amino acids having a sequence derived from a human V_(κ) L15germline sequence; (d) a heavy chain variable region that comprisesconsecutively linked amino acids having a sequence derived from a humanV_(H) 1-69 germline sequence, and a light chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(κ) A27 germline sequence; (e) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 3-9 germline sequence, and a light chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(κ) L15 germline sequence; or (f) aheavy chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(H) 3-9 germlinesequence, and a light chain variable region that comprises consecutivelylinked amino acids having a sequence derived from a human V_(κ) L18germline sequence.

An example of an Ab having a V_(H) and a V_(κ) derived from V_(H) 1-18and V_(κ) L6 germline sequences, respectively, is 3G10. Examples of Abshaving V_(H) and V_(κ) regions derived from V_(H) 1-69 and V_(κ) L6germline sequences, respectively, include 12A4, 1B12, 7H1 and 12B7. Anexample of an Ab having a V_(H) and a V_(κ) derived from V_(H) 1-3 andV_(κ) L15 germline sequences, respectively, is 10A5. Examples of Abshaving V_(H) and V_(κ) regions derived from V_(H) 1-69 and V_(κ) A27germline sequences, respectively, include 5F8, 11E6 and 11E6a. Anexample of an Ab having a V_(H) and a V_(K) derived from V_(H) 3-9 andV_(κ) L15 germline sequences, respectively, is 10H10. An example of anAb having a V_(H) and a V_(κ) derived from V_(H) 1-3 and V_(κ) L15germline sequences, respectively, is 10A5. An example of an Ab having aV_(H) and a V_(κ) derived from V_(H) 3-9 and V_(κ) L18 germlinesequences, respectively, is 13G4.

In certain embodiments, anti-PD-L1 Abs of the invention comprise heavyand light chain variable regions having amino acid sequences that arehighly similar or homologous to the amino acid sequences of thepreferred anti-PD-L1 Abs described herein, wherein the Ab retains thefunctional properties of the aforementioned anti-PD-L1 Abs of theinvention. For example, Abs of the invention include mAbs comprising aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises consecutively linked aminoacids having a sequence that is at least 80% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs. 15, 16, 17,18, 19, 20, 21, 22, 23, and 24, and the light chain variable regioncomprises consecutively linked amino acids having a sequence that is atleast 80% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34. Inother embodiments, the V_(H) and/or V_(L) amino acid sequences mayexhibit at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to thesequences set forth above.

Certain embodiments of the anti-PD-L1 Abs of the invention compriseheavy and light chain variable regions each comprising CDR1, CDR2 andCDR3 domains, wherein one or more of these CDR domains compriseconsecutively linked amino acids having sequences that are the same asthe CDR sequences of the preferred anti-PD-L1 Abs described herein(e.g., 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4), orconservative modifications thereof, and wherein the Abs retain thedesired functional properties of the preferred anti-PD-L1 Abs of theinvention.

On the basis of the evidence that the heavy chain CDR3 is the primarydeterminant of binding specificity and affinity of an Ab, it isgenerally true that once the heavy chain CDR3 sequence of a given Ab isdefined, variability in the other five CDR sequences does not greatlyaffect the binding specificity of that Ab. Accordingly, anti-PD-L1 Absof the invention include isolated Abs comprising 6 CDRs, wherein the Absare defined by specifying the sequence of the heavy chain CDR3 domain.

Anti-PD-L1 Abs of the invention also include isolated Abs that bindspecifically to human PD-L1 and cross-compete for binding to human PD-L1with any of HuMAbs 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7and 13G4. Thus, anti-PD-L1 Abs of the invention include isolated Abs orantigen-binding portions thereof that cross-compete for binding to PD-L1with a reference Ab or a reference antigen-binding portion thereofcomprising: (a) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 15 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 25; (b) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 16 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 26; (c) a human heavy chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 17 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 27; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 18 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 28; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 19 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 29; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 20 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 30; (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 21 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEO ID NO: 31; (h) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 22 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 32; (i) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 23 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEO ID NO: 33; or (j) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 24 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 34.

The ability of an Ab to cross-compete with any of 3G10, 12A4, 10A5, 5F8,10H10, 1B12, 7H1, 11E6, 12B7 and 13G4 for binding to human PD-L1demonstrates that such Ab binds to the same epitope region of each of3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4,respectively. All isolated Abs that bind to the same epitope region ofhuman PD-L1 as does HuMAb 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6,12B7 or 13G4 are included among the Abs of the invention. Thesecross-competing Abs are expected to have very similar functionalproperties by virtue of their binding to the same epitope region ofPD-L1. For example, cross-competing anti-PD-L1 mAbs 3G10, 1B12, 13G4,12A4 (BMS-936559), 10A5, 12B7, 11E6 and 5F8 have been shown to havesimilar functional properties (see U.S. Pat. No. 7,943,743 at Examples3-11), whereas mAb 10H10, which binds to a different epitope region,behaves differently (U.S. Pat. No. 7,943,743 at Example 11). The higherthe degree of cross-competition, the more similar will the functionalproperties be. Further, cross-competing Abs can be identified instandard PD-L1 binding assays, e.g., BIACORE® analysis, ELISA assays orflow cytometry, that are well known to persons skilled in the art. Inpreferred embodiments, the Abs that cross-compete for binding to humanPD-1 with, or bind to the same epitope region of human PD-L1 as, 3G10,12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4 are mAbs,preferably chimeric Abs, or more preferably humanized or human Abs. Suchhuman mAbs can be prepared and isolated as described in U.S. Pat. No.7,943,743.

Data provided in Example 2 show that each of the anti-PD-L1 HuMAbs 5F8,7H1, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7 and 13G4, i.e., all of theHuMAbs tested except 10H10, substantially blocked binding of mAbs 3G10,10A5, 11E6, 12A4 and 13G4 to Chinese Hamster Ovary (CHO) cellsexpressing PD-L1 cells. HuMAb 10H10 substantially blocked the bindingonly of itself to CHO/PD-L1 cells. These data show that 3G10, 10A5,11E6, 12A4 and 13G4 cross-compete with all of the HuMAbs tested, exceptfor 10H10, for binding to the same epitope region of human PD-L1 (FIGS.2A-F).

Data provided in Example 3 show that the binding of HuMAb 12A4 to ES-2ovarian carcinoma cells expressing PD-L1 cells was substantially blockedby 12A4 itself and by 1B12 and 12B7, and was moderately to significantlyblocked by mAbs 5F8, 10A5, 13G4 and 3G10, but was not blocked by mAb10H10. These data, largely consistent with the data in Example 2, showthat 12A4 itself, and 2 other HuMabs, 12B7 and 1B12, substantiallycross-compete with 12A4 for binding to the same epitope region, possiblythe same epitope, of human PD-L1; 5F8, 10A5, 13G4 and 3G10, exhibit asignificant but lower level of cross-competition with 12A4, suggestingthat these mAbs may bind to epitopes that overlap the 12A4 epitope;whereas 10H10 does not cross-compete at all with 12A4 (FIG. 3),suggesting that this mAb binds to a different epitope region from 12A4.

Anti-PD-L1 Abs of the invention also include Abs engineered startingfrom Abs having one or more of the V_(H) and/or V_(L) sequencesdisclosed herein, which engineered Abs may have altered properties fromthe starting Abs. An anti-PD-L1 Ab can be engineered by a variety ofmodifications as described above for the engineering of modifiedanti-PD-1 Abs of the invention.

Anti-PD-L1 Abs of the invention also include isolated Abs selected fortheir ability to bind to PD-L1 in formalin-fixed, paraffin-embedded(FFPE) tissue specimens. The use of FFPE samples is essential for thelong-term follow-up analysis of the correlation between PD-L1 expressionin tumors and disease prognosis or progression. Yet, studies onmeasuring PD-L1 expression have often been conducted on frozen specimensbecause of the difficulty in isolating anti-human PD-L1 Abs that can beused to stain PD-L1 in FFPE specimens by IHC in general (Hamanishi etal., 2007) and, in particular, Abs that bind specifically to membranousPD-L1 in these tissues. The use of different Abs to stain PD-L1 infrozen versus FFPE tissues, and the ability of certain Abs todistinguish membranous and/or cytoplasmic forms of PD-L1, may accountfor some of the disparate data reported in the literature correlatingPD-L1 expression with disease prognosis (Hamanishi et al., 2007; Gadiotet al., 2011). This disclosure provides several rabbit mAbs that bindwith high affinity specifically to membranous human PD-L1 in FFPE tissuesamples comprising tumor cells and tumor-infiltrating inflammatorycells.

Rabbit and mouse anti-hPD-L1 mAbs were produced as described in theExamples. Out of almost 200 rabbit Ab multiclones and purified mousesubclones screened, only ten rabbit multiclone Abs were found tospecifically detect the membranous form of PD-L1, and the top fivemulticlones (designated Nos. 13, 20, 28, 29 and 49) were subsequentlysubcloned. The clone that produced the most robust detectionspecifically of membranous PD-L1, rabbit clone 28-8, was selected forthe IHC assays. The sequences of the variable regions of mAb 28-8 areset forth in SEQ ID NOs. 35 and 36, respectively. Rabbit clones 28-1,28-12, 29-8 and 20-12 were the next best mAbs in terms of robustdetection of membranous PD-L1 in FFPE tissues.

Anti-PD-L1 Abs of the invention also include antigen-binding portions ofthe above Abs, including Fab, F(ab′)₂ Fd, Fv, and scFv, di-scFv orbi-scFv, and scFv-Fc fragments, diabodies, triabodies, tetrabodies, andisolated CDRs (see Hollinger et al., 2005; Olafsen et al., 2010, forfurther details).

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the present disclosure pertains to isolated nucleicacid molecules that encode any of the Abs of the invention. Thesenucleic acids may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. A nucleic acid of theinvention can be, for example, DNA or RNA, and may or may not containintronic sequences. In a preferred embodiment, the nucleic acid is acDNA.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For Abs expressed by hybridomas (e.g., hybridomasprepared from transgenic mice carrying human immunoglobulin genes asdescribed further below), cDNAs encoding the light and heavy chains ofthe Ab made by the hybridoma can be obtained by standard PCRamplification or cDNA cloning techniques. Nucleic acids encoding Absobtained from an immunoglobulin gene library (e.g., using phage displaytechniques) can be recovered from the library.

Preferred nucleic acids molecules of the invention are those encodingthe V_(H) and V_(κ) sequences of the anti-PD-1 HuMAbs, 17D8, 2D3, 4H1,5C4, 4A11, 7D3 and 5F4 (disclosed in U.S. Pat. No. 8,008,449), and thoseencoding the V_(H) and V_(κ) sequences of the anti-PD-L1 HuMAbs, 3G10,12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 (disclosed inU.S. Pat. No. 7,943,743). An isolated DNA encoding the V_(H) region canbe converted to a full-length heavy chain gene by operatively linkingthe V_(H)-encoding DNA to another DNA molecule encoding heavy chainconstant regions (C_(H1)/C_(H2) and C_(H3)), the sequences of which areknown in the art and can be obtained by standard PCR amplification. Theheavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE,IgM or IgD constant region, but is preferably an IgG1 or IgG4 constantregion. Similarly, an isolated DNA encoding the V_(L) region can beconverted to a full-length light chain gene by operatively linking theV_(L)-encoding DNA to another DNA molecule encoding the light chainconstant region (C_(L)), the sequence of which is known in the art andcan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or lambda constant region, but most preferably isa kappa constant region.

Pharmaceutical Compositions

Antibodies of the present invention may be constituted in a composition,e.g., a pharmaceutical composition, containing one Ab or a combinationof Abs, or an antigen-binding portion(s) thereof, and a pharmaceuticallyacceptable carrier. As used herein, a “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Preferably,the carrier is suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g., by injection orinfusion). A pharmaceutical composition of the invention may include oneor more pharmaceutically acceptable salts, anti-oxidant, aqueous andnonaqueous carriers, and/or adjuvants such as preservatives, wettingagents, emulsifying agents and dispersing agents.

Dosage regimens are adjusted to provide the optimum desired response,e.g., a therapeutic response or minimal adverse effects. Foradministration of an anti-PD-1 or anti-PD-L1 Ab, the dosage ranges fromabout 0.0001 to about 100 mg/kg, usually from about 0.001 to about 20mg/kg, and more usually from about 0.01 to about 10 mg/kg, of thesubject's body weight. Preferably, the dosage is within the range of0.1-10 mg/kg body weight. For example, dosages can be 0.1, 0.3, 1, 3, 5or 10 mg/kg body weight, and more preferably, 0.3, 1, 3, or 10 mg/kgbody weight. The dosing schedule is typically designed to achieveexposures that result in sustained receptor occupancy (RO) based ontypical pharmacokinetic properties of an Ab. An exemplary treatmentregime entails administration once per week, once every two weeks, onceevery three weeks, once every four weeks, once a month, once every 3months or once every three to 6 months. The dosage and scheduling maychange during a course of treatment. For example, dosing schedule maycomprise administering the Ab: (i) every two weeks in 6-week cycles;(ii) every four weeks for six dosages, then every three months; (iii)every three weeks; (iv) 3-10 mg/kg body weight once followed by 1 mg/kgbody weight every 2-3 weeks. Considering that an IgG4 Ab typically has ahalf-life of 2-3 weeks, a preferred dosage regimen for an anti-PD-1 oranti-PD-L1 Ab of the invention comprises 0.3-10 mg/kg body weight,preferably 3-10 mg/kg body weight, more preferably 3 mg/kg body weightvia intravenous administration, with the Ab being given every 14 days inup to 6-week or 12-week cycles until complete response or confirmedprogressive disease.

In some methods, two or more mAbs with different binding specificitiesare administered simultaneously, in which case the dosage of each Abadministered falls within the ranges indicated. Antibody is usuallyadministered on multiple occasions. Intervals between single dosages canbe, for example, weekly, every 2 weeks, every 3 weeks, monthly, everythree months or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of Ab to the target antigen in the patient. Insome methods, dosage is adjusted to achieve a plasma Ab concentration ofabout 1-1000 μg/ml and in some methods about 25-300 μg/ml.

Alternatively, the Ab can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the Ab in thepatient. In general, human Abs show the longest half-life, followed byhumanized Abs, chimeric Abs, and nonhuman Abs. The dosage and frequencyof administration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being unduly toxic to the patient. Theselected dosage level will depend upon a variety of pharmacokineticfactors including the activity of the particular compositions of thepresent invention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts. A composition of the present invention can be administeredvia one or more routes of administration using one or more of a varietyof methods well known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

Uses and Methods of the Invention

The Abs, Ab compositions, nucleic acids and methods of the presentinvention have numerous in vitro and in vivo utilities including, forexample, methods to determine and quantify the expression of PD-1 orPD-L1 comprising binding of the Abs to the target polypeptides ormeasuring the amount of nucleic acid encoding these polypeptides, and amethod for immunotherapy of a subject afflicted with a diseasecomprising administering to the subject a composition comprising atherapeutically effective amount of a therapeutic agent that inhibitssignaling from an inhibitory immunoregulator. In preferred embodimentsof the latter method, the inhibitory immunoregulator is a component ofthe PD-1/PD-L1 signaling pathway, and the therapeutic agent disruptssignaling of this pathway. More preferably, the therapeutic agent is anAb that interferes with the interaction between PD-1 and PD-L1. Incertain preferred embodiments of this method, the Ab binds specificallyto PD-1 and blocks the interaction of PD-1 with PD-L1 and/or PD-L2. Inother preferred embodiments, the therapeutic agent is an Ab that bindsspecifically to PD-L1 and blocks the interaction of PD-L1 with PD-1and/or B7-1 (CD80). Thus, the disclosure provides methods forpotentiating an immune response in a subject comprising administering ananti-PD-1 and/or an anti-PD-L1 Ab in order to disrupt the interactionbetween PD-1 and PD-L1, and methods of treating diseases mediated bysuch a potentiation of the immune response. When Abs to PD-1 and PD-L1are administered together, the two can be administered sequentially ineither order or simultaneously. In certain aspects, this disclosureprovides methods of modifying an immune response in a subject comprisingadministering to the subject an anti-PD-1 and/or an anti-PD-L1 Ab of theinvention, or antigen-binding portion thereof, such that the immuneresponse in the subject is modified. Preferably, the immune response ispotentiated, enhanced, stimulated or up-regulated. In preferredembodiments, the Abs of the present invention are human Abs.

Preferred subjects include human patients in need of enhancement of animmune response. The immunotherapeutic methods disclosed herein areparticularly suitable for treating human patients having a disorder thatcan be treated by potentiating a T-cell mediated immune response. Incertain embodiments, the methods are employed for treatment of subjectsafflicted with a disease caused by an infectious agent. In preferredembodiments, the methods are employed for treatment of subjectsafflicted with, or at risk of being afflicted with, a cancer.

Cancer Immunotherapy

Blockade of PD-1/PD-L1 interaction has been shown to potentiate immuneresponses in vitro (U.S. Pat. Nos. 8,008,449 and 7,943,743; Fife et al.,2009) and mediate preclinical antitumor activity (Dong et al., 2002;Iwai et al., 2002). However, the molecular interactions potentiallyblocked by these two Abs are not identical: anti-PD-1 Abs of theinvention disrupt PD-1/PD-L1 and potentially PD-1/PD-L2 interactions; incontrast, whereas anti-PD-L1 Abs of the invention also disruptPD-1/PD-L1 interactions, they do not block PD-1/PD-L2 interactions butinstead may disrupt the PD-1-independent PD-L1/CD80 interaction, whichhas also been shown to down-modulate T-cell responses in vitro and invivo (Park et al., 2010; Paterson et al., 2011; Yang et al., 2011; Butteet al., 2007; Butte et al., 2008). Thus, it is possible that among thesevaried ligand-receptor pairings, different interactions may dominate indifferent cancer types, contributing to dissimilar activity profiles forthe two Abs.

Disruption of the PD-1/PD-L1 interaction by antagonistic Abs can enhancethe immune response to cancerous cells in a patient. PD-L1 is notexpressed in normal human cells, but is abundant in a variety of humancancers (Dong et al., 2002). The interaction between PD-1 and PD-L1impairs T cell responses as manifested by a decrease intumor-infiltrating lymphocytes (TILs) and a decrease in T-cell receptormediated proliferation, resulting in T cell anergy, exhaustion orapoptosis, and immune evasion by the cancerous cells (Zou et al., 2008;Blank et al., 2005; Konishi et al., 2004; Dong et al., 2003; Iwai etal., 2002) Immune suppression can be reversed by inhibiting the localinteraction between PD-L1 and PD-1 using an anti-PD-1 and/or ananti-PD-L1 Ab. These Abs may be used alone or in combination to inhibitthe growth of cancerous tumors. In addition, either or both of these Absmay be used in conjunction with other immunogenic agents includingcytokines, standard cancer chemotherapies, vaccines, radiation, surgery,or other Abs.

Immunotherapy of Cancer Patients Using an Anti-PD-1 Antibody

This disclosure provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises administering to thesubject a composition comprising a therapeutically effective amount ofan Ab or an antigen-binding portion thereof that disrupts theinteraction of PD-1 with PD-L1 and/or PD-L2. The disclosure alsoprovides a method of inhibiting growth of tumor cells in a subject,comprising administering to the subject an Ab or an antigen-bindingportion thereof that disrupts the interaction of PD-1 with PD-L1 and/orPD-L2 in an amount effective to inhibit growth of the tumor cells. Inpreferred embodiments, the subject is a human. In other preferredembodiments, the Ab or antigen-binding portion thereof is an anti-PD-1Ab of the invention or an antigen-binding portion thereof. In certainembodiments, the Ab or antigen-binding portion thereof is of an IgG1 orIgG4 isotype. In certain embodiments, the Ab or antigen-binding portionthereof is a mAb or an antigen-binding portion thereof. In certain otherembodiments, the Ab or antigen-binding portion thereof is a chimeric,humanized or human Ab or an antigen-binding portion thereof. Inpreferred embodiments for treating human subjects, the Ab orantigen-binding portion thereof is a human Ab or an antigen-bindingportion thereof.

The clinical trials described in the Examples employed the anti-PD-1HuMAb, nivolumab (designated 5C4 in U.S. Pat. No. 8,008,449), to treatcancer. While 5C4 was selected as the lead Ab for entering the clinic,it is notable that several anti-PD-1 Abs of the invention share with 5C4functional properties that are important to the therapeutic activity of5C4, including high affinity binding specifically to human PD-1,increasing T-cell proliferation, IL-2 secretion and interferon-γproduction in an MLR assay, inhibiting the binding of PD-L1 and/or PD-L2to PD-1, and inhibiting tumor cell growth in vivo. Moreover, certain ofthe anti-PD-1 Abs of the invention, 17D8, 2D3, 4H1 and 7D3 arestructurally related to 5C4 in comprising V_(H) and V_(κ) regions thathave sequences derived from V_(H) 3-33 and V_(κ) L6 germline sequences,respectively. In addition, 5C4, 2D3, 7D3, 4H1 and 17D8 all cross-competefor binding to the same epitope region of hPD-1 (Example 1). Thus, thepreclinical characterization of nivolumab and other anti-PD-1 HuMabsindicate that the methods of treating cancer provided herein may beperformed using different Abs selected from the broad genus of anti-PD-1Abs of the invention.

Accordingly, certain embodiments of the immunotherapy methods disclosedherein comprise administering to a patient an anti-PD-1 Ab orantigen-binding portion thereof comprising: (a) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 3-33 germline sequence, and a light chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(κ) L6 germline sequence, or (b) a heavychain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(H) 4-39 germline sequence, anda light chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L15 germlinesequence.

In certain other embodiments, the Ab or antigen-binding portion thereofthat is administered to the patient cross-competes for binding to PD-1with a reference Ab or a reference antigen-binding portion thereofcomprising: (a) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 1 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 8; (b) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 2 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 9; (c) a human heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 3 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 10; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 4 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 11; (e) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 5 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 12; (f) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 6 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 13; or (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 7 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEO IDNO: 14. In preferred embodiments, the Ab or antigen-binding portionthereof cross-competes for binding to PD-1 with nivolumab.

In certain preferred embodiments of the immunotherapy methods disclosedherein, the anti-PD-1 Ab or antigen-binding portion thereof administeredto the patient comprises: (a) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 1 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 8; (b) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 2 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 9; (c) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 3 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 10; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 4 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 11; (e) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 5 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 12; (f) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 6 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 13; or (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 7 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEO IDNO: 14. In more preferred embodiments, the anti-PD-1 Ab orantigen-binding portion comprises a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 4 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 11. In yet more preferred embodiments, the anti-PD-1 Ab isnivolumab.

In the clinical trials of anti-PD-1 immunotherapy described in theExamples below, intriguing ORs with durable clinical responses, even inheavily pretreated patients, were observed across multiple tumor typesincluding a substantial proportion of NSCLC, MEL, and RCC patients andin various sites of metastasis including liver, lung, lymph nodes, andbone. See, also, Topalian et al. (2012b). MEL and RCC are considered tobe immunogenic neoplasms, having previously been demonstrated to beresponsive to cancer immunotherapy, e.g., IL-2 (McDermott et al., 2006)and/or anti-CTLA-4 Ab (Hodi et al., 2010). In contrast, NSCLC has beenconsidered “non-immunogenic” and poorly responsive to immune-basedtherapies (Holt et al., 2011). Thus, the results with NSCLC areparticularly striking, unexpected and surprising. In NSCLC patients,based on data analyzed up to February 2012, 14 ORs were observed atBMS-936558 doses of 1, 3, or 10 mg/kg with response rates of 6%, 32%,and 18%, respectively. ORs were observed across NSCLC histologies: 6responders of 18 squamous (33%), 7 of 56 nonsquamous (13%), and 1 of 2unknown. This level of activity seen with anti-PD-1 in NSCLC patientswith significant prior therapy (47% with 3 lines of previous therapy)and across histologies is unique, particularly in the squamous histologypatients (cf. Gridelli et al., 2008; Miller, 2006), and provides a veryfavorable benefit/risk dynamic regarding efficacy and safety compared toexisting standard-of-care.

Durability of Clinical Responses to Anti-PD-1 in Heavily PretreatedCancer Patients

The durability of ORs across multiple cancer types in patients treatedwith the anti-PD-1 Ab is particularly notable. The objective responserate (ORR) in heavily pretreated NSCLC patients receiving anti-PD-1 Ab,including patients with squamous histology, is particularly surprisingand unexpected, as standard salvage therapies historically show modestbenefit in these patients (Scagliotti et al., 2011). As measured bystandard RECIST in this study, ORs were long-lasting, with responsedurations ≥1 year in 20 of 31 responders in the data analyzed up toFebruary 2012. In addition, patterns of tumor regression consistent withimmune-related patterns of response were observed.

These findings have established the PD-1 pathway as a new therapeuticfocus in oncology (Pardoll, 2012; Topalian et al., 2012c; McDermott etal., 2013). In the current study, in which 47% of patients hadprogressive disease following 3 or more prior systemic regimens,preliminary analysis has been conducted up to March 2013. This updatedanalysis has supported and reinforced the data obtained and conclusionsreached from the earlier February 2012 analyses. Thus, conventional ORswere documented in patients with NSCLC (16%), MEL (31%) and RCC (29%),and prolonged disease stabilization in others (9%, 6%, and 27%,respectively) across all doses tested (see Example 7, Table 2).Additionally, 13 patients (4%) manifested unconventional,“immune-related” response patterns as previously described withanti-CTLA-4 therapy, several of which were sustained (Sharma et al.,2011). The updated analyses again underscored the durability of survivalin nivolumab-treated patients, which has not been observed withchemotherapy or small molecule inhibitors to date, but has been observedin patients with advanced melanoma receiving ipilimumab, another immunecheckpoint blocking agent (Hodi et al., 2010).

Of particular importance, the objective tumor regression and diseasestabilization induced by nivolumab in heavily-pretreated patients withadvanced NSCLC, MEL, and RCC translate to survival outcomes that comparevery favorably with historical data for these patient populationstreated with conventional chemotherapy and/or tyrosine-kinase inhibitor(TKI) treatments. In NSCLC, nivolumab induced median overall survivalsof 9.6 and 9.2 months in patients with squamous and non-squamoushistologies, respectively. Landmark survival rates of 43% (1-year), 32%(2-year) and 24% (3-year) were achieved (see Example 7, Table 2). Thishigh level of efficacy is especially impressive since 55% of thesepatients had received 3 or more prior therapies. Historically, 2 Lchemotherapeutics for lung cancer (i.e., docetaxel and pemetrexed) haveachieved a median overall survival of 7.5-8.3 months (Shepherd et al.,2000; Hanna et al., 2004). In a ⅔-L population, erlotinib-treatedpatients had a median survival of 6.7 months, versus 4.7 months inplacebo-treated patients (Shepherd et al., 2005). No therapy iscurrently approved for use in lung cancer beyond the 3 L setting, andminimal data exist to benchmark survival in this patient population.

In nivolumab-treated MEL patients, overall survival (OS) of 16.8 monthswas achieved, with landmark survival rates of 61% (1-year), 44% (2-year)and 40% (3-year) (see Example 7, Table 2). Survival outcomes inpretreated melanoma patients supported the recent PDA approvals ofipilimumab and vemurafenib. In patients with at least one priortreatment for metastatic disease, ipilimumab increased median OS from6.4 to 10.1 months, compared to a gp100 peptide vaccine (Hodi et al.,2010). In phase 2 trials of ipilimumab in previously treated patients,landmark 2-year survival rates ranged from 24.2-32.8% (Lebbe et al.,2012). Median OS in previously treated MEL patients enrolled on a largephase 2 of vemurafenib was 15.9 months (Sosman et al., 2012).

In nivolumab-treated patients with RCC, among whom 44% received 3 ormore prior therapies and 74% received prior anti-angiogenic therapy, themedian OS has not been achieved and exceeds 22 months. Landmark survivalrates of 70% (1-year), 52% (2-year) and 52% (3-year) were achieved (seeExample 7, Table 2). In a recent Phase 3 trial enrolling kidney cancerpatients whose disease progressed following anti-angiogenic therapy,everolimus was compared with placebo: median OS was 14.8 versus 14.4months, respectively (Motzer et al., 2008; Motzer et al., 2008). Arecent Phase 3 trial comparing sorafenib to temsirolimus in asunitinib-refractory kidney cancer population yielded median OS of 16.6and 12.3 months, respectively (Hutson et al., 2012). Thus, treatment ofa heavily-pretreated patient population with nivolumab has yielded aconsiderably longer median OS (>22 months) than treatment of a lessrefractory population with standard-of-care therapies. Controlled Phase3 trials with prospective survival endpoints are underway in NSCLC, MELand RCC (NCT01673867, NCT01721772, NCT01642004, NCT01668784, andNCT01721746 (see Clinical Trials Website). The results from these trialsare expected to further demonstrate the high efficacy of, and durabilityof responses to, nivolumab in these cancers compared standard-of-caretherapies.

The data disclosed herein demonstrating the high efficacy, durabilityand broad applicability of anti-PD-1 immunotherapy for treating cancerhas led to nivolumab being tested for additional types of cancer. Forexample, on the basis that increased PD-L1 expression has been reportedwith various hematologic malignancies and may prevent the host immuneresponse from exerting a beneficial impact on the malignant cells, atrial to confirm the ability of nivolumab to mediate antitumor activityin patients with hematologic malignancies (multiple myeloma, B-celllymphoma, T-cell lymphoma, Hodgkin's lymphoma/primary mediastinal B-celllymphoma, and chronic myelogenous leukemia) has been initiated(NCT01592370). Nivolumab is also being tested as a monotherapy inadvanced hepatocellular carcinoma (NCT01658878).

In summary, the results of anti-PD-1 immunotherapy disclosed herein areremarkable in at least the following three respects. First, anti-PD-1has been shown to be highly efficacious compared to historical data forpatients on standard-of-care treatments for cancer. Notably, thisefficacy has been demonstrated in patient in heavily pretreatedpopulations in which about half of the patients had progressive diseasefollowing 3 or more prior systemic regimens. Such patients, afflictedwith advanced, metastatic and/or refractory cancers, are notoriouslydifficult to treat. Accordingly, this disclosure provides methods forimmunotherapy of a patient afflicted with an advanced, metastatic and/orrefractory cancer, which method comprises administering to the patient atherapeutically effective amount of an Ab or an antigen-binding portionthereof that disrupts the interaction of PD-1 with PD-L1 and/or PD-L2.In certain embodiments of any of the therapeutic methods disclosedherein, the subject has been pre-treated for the cancer; for example,the subject had undergone at least one, two, or three prior lines oftherapy for cancer.

Second, the present therapeutic methods have been shown to be applicableto a broad genus of different cancers. Based on the surprising discoverythat even a “non-immunogenic” cancer such as NSCLC (Holt et al., 2011)and hard-to-treat cancers such as ovarian and gastric cancers (as wellas other cancers tested, including MEL, RCC, and CRC) are amendable totreatment with anti-PD-1 and/or anti-PD-L1 (see Examples 7 and 14), thisdisclosure provides methods for immunotherapy of a patient afflictedwith any cancer.

Third, treatment with an anti-PD-1 or anti-PD-L1 Ab has been shown toproduce strikingly durable clinical activity in cancer patients.Accordingly, this disclosure provides immunotherapeutic methods ofinducing a durable clinical response in a cancer patient comprisingadministering to the patient a therapeutically effective amount of an Abor an antigen-binding portion thereof that disrupts the interaction ofPD-1 with PD-L1 and/or PD-L2. In preferred embodiments of any of thetherapeutic methods described herein, the clinical response is a durableresponse.

As used herein, a “durable” response is a therapeutic or clinicalresponse that exceeds the anticipated median OS rate in a patientpopulation. The anticipated median OS rate varies with different cancerand different patient populations. In certain embodiments, a durableresponse exceeds the anticipated median OS rate in the relevant patientpopulation by at least 10%, preferably by at least 20%, more preferablyby at least 30%, and even more preferably by at least 50%. A majorbenefit of immunotherapeutic approaches based on PD-1 pathway blockademay be the functional restoration of exhausted T cells with long-termgeneration of memory T cells that may maintain antitumor immunesurveillance and inhibit tumor growth for prolonged periods extending tomany years, even in the absence of continued therapy (Kim et al., 2010).Indeed, long-term follow-up studies on patients following cessation ofnivolumab therapy have confirmed that a patient with CRC experienced acomplete response which was ongoing after 3 years; a patient with RCCexperienced a partial response lasting 3 years off therapy, whichconverted to a complete response that was ongoing at 12 months; and apatient with melanoma achieved a partial response that was stable for 16months off therapy, and recurrent disease was successfully treated withreinduction anti-PD-1 therapy (Lipson et al., 2013).

Combination Therapy Including Anti-PD-1 Abs

While monotherapy with anti-PD-1 and anti-PD-L1 Abs has been shownherein to significantly increase the survival of patients with lungcancer, melanoma, kidney cancer, and potentially other malignancies,preclinical evidence indicates that synergistic treatment combinationsbased on PD-1 pathway blockade could have even more potent effects.Clinical evaluation of nivolumab combined with ipilimumab (anti-CTLA-4),whose mechanism of action is similar yet distinct from nivolumab's(Mellman et al., 2011; Topalian et al., 2012c), is ongoing (Wolchok etal., 2013), as are studies of nivolumab in combination with melanomavaccines (NCT01176461, NCT01176474; Weber et al., 2013), and BMS-986015,an anti-KIR Ab (NCT01714739).

Anti-PD-1 Abs can be combined with an immunogenic agent, for example apreparation of cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules),antigen-presenting cells such as dendritic cells bearingtumor-associated antigens, and/or cells transfected with genes encodingimmune stimulating cytokines (He et al., 2004). Non-limiting examples oftumor vaccines that can be used include peptides of melanoma antigens,such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF.PD-1 blockade may also be effectively combined with standard cancertreatments, including chemotherapeutic regimes, radiation, surgery,hormone deprivation and angiogenesis inhibitors, as well as anotherimmunotherapeutic Ab (e.g., an anti-PD-L1, anti-CTLA-4 and/or anti-LAG-3Ab).

Immune-Related Clinical Responses

It has become evident that conventional response criteria may notadequately assess the activity of immunotherapeutic agents becauseprogressive disease (by initial radiographic evaluation) does notnecessarily reflect therapeutic failure. For example, treatment with theanti-CTLA-4 Ab, ipilimumab, has been shown to produce four distinctresponse patterns, all of which were associated with favorable survival:(a) shrinkage in baseline lesions, without new lesions; (b) durablestable disease (in some patients followed by a slow, steady decline intotal tumor burden); (c) response after an increase in total tumorburden; and (d) response in the presence of new lesions. Accordingly, toproperly evaluate immunotherapeutic agents, long-term effects on thetarget disease must also be captured. In this regard, systematicimmune-related response criteria (irRC) that make allowances for anearly increase in tumor burden and/or the appearance of new lesions, andwhich seek to enhance the characterization of immune-related responsepatterns, have been proposed (Wolchok et al., 2009). While the fullimpact of these unconventional response patterns remains to be definedin randomized trials of nivolumab with survival endpoints, the presentobservations are reminiscent of findings with ipilimumab in which asignificant extension of OS was observed in treated patients (Hodi etal., 2010; Robert et al., 2011).

The overall risk/benefit profile of anti-PD-1 immunotherapy is alsofavorable, with a low incidence of more severe drug-related adverseevents (AEs; >grade 3), the specific events observed to date beingconsistent with other immunotherapeutic agents. This suggests thatanti-PD-1 immunotherapy can be delivered in an outpatient setting withminimal supportive care.

Broad Spectrum of Cancers Treatable by Anti-PD-1 Immunotherapy

The clinical data presented herein demonstrate that immunotherapy basedon PD-1 blockade is not limited to only “immunogenic” tumor types, suchas MEL and RCC, but extends to tumor types not generally considered tobe immune-responsive, including NSCLC. The unexpected successes withtreatment-refractory metastatic NSCLC underscore the possibility thatany neoplasm can be “immunogenic” in the context of proper immunemodulation, and suggest that PD-1 blockade as an immunotherapeuticapproach is broadly applicable across a very diverse range of tumortypes. Cancers that may be treated using the anti-PD-1 Abs of theinvention also include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment include NSCLC,MEL, RCC, CRC, CRPC, HCC, squamous cell carcinoma of the head and neck,carcinomas of the esophagus, ovary, gastrointestinal tract and breast,and a hematologic malignancy. Although NSCLC is not generally consideredresponsive to immunotherapy, data disclosed herein unexpectedlydemonstrate that both squamous and non-squamous NSCLC are responsive totreatment with an anti-PD-1 Ab. Additionally, the disclosure providesfor the treatment of refractory or recurrent malignancies whose growthmay be inhibited using an anti-PD-1 Ab of the invention.

Examples of other cancers that may be treated using an anti-PD-1 Ab inthe methods of the present invention, based on the indications of verybroad applicability of anti-PD-1 immunotherapy provided herein, includeliver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of thehead or neck, breast cancer, lung cancer, cutaneous or intraocularmalignant melanoma, renal cancer, uterine cancer, ovarian cancer,colorectal cancer, colon cancer, rectal cancer, cancer of the analregion, stomach cancer, testicular cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, environmentally induced cancersincluding those induced by asbestos, hematologic malignancies including,for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primarymediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloidlymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia,follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma,immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides,anaplastic large cell lymphoma, T-cell lymphoma, and precursorT-lymphoblastic lymphoma, and any combinations of said cancers. Thepresent invention is also applicable to treatment of metastatic cancers.

Medical Uses of Anti-PD-1 Abs

One aspect of this invention is the use of any anti-PD-1 Ab orantigen-binding portion thereof of the invention for the preparation ofa medicament for inhibiting signaling from the PD-1/PD-L1 pathway so asto thereby potentiate an endogenous immune response in a subjectafflicted with cancer. Another aspect is the use of any anti-PD-1 Ab oran antigen-binding portion thereof of the invention for the preparationof a medicament for immunotherapy of a subject afflicted with cancercomprising disrupting the interaction between PD-1 and PD-L1. These usesfor the preparation of medicaments are broadly applicable to the fullrange of cancers disclosed herein. In preferred embodiments of theseuses, the cancers include squamous NSCLC, non-squamous NSCLC, MEL, RCC,CRC, CRPC, HCC, squamous cell carcinoma of the head and neck, andcarcinomas of the esophagus, ovary, gastrointestinal tract and breast,and a hematologic malignancy. This disclosure also provides medical usesof any anti-PD-1 Ab or antigen-binding portion thereof of the inventioncorresponding to all the embodiments of the methods of treatmentemploying an anti-PD-1 Ab described herein.

The disclosure also provides an anti-PD-1 Ab or an antigen-bindingportion thereof of the invention for use in potentiating an endogenousimmune response in a subject afflicted with cancer by inhibitingsignaling from the PD-1/PD-L1 pathway. The disclosure further providesan anti-PD-1 Ab or an antigen-binding portion thereof of the inventionfor use in immunotherapy of a subject afflicted with cancer comprisingdisrupting the interaction between PD-1 and PD-L1. These Abs may be usedin potentiating an endogenous immune response against, or inimmunotherapy of, the full range of cancers disclosed herein. Inpreferred embodiments, the cancers include squamous NSCLC, non-squamousNSCLC, MEL, RCC, CRC, CRPC, HCC, squamous cell carcinoma of the head andneck, and carcinomas of the esophagus, ovary, gastrointestinal tract andbreast, and a hematologic malignancy.

Immunotherapy of Cancer Patients Using an Anti-PD-L1 Antibody

PD-L1 is the primary PD-1 ligand up-regulated within solid tumors, whereit can inhibit cytokine production and the cytolytic activity ofPD-1-positive, tumor-infiltrating CD4⁺ and CD8⁺ T-cells, respectively(Dong et al., 2002; Hino et al., 2010; Taube et al., 2012). Theseproperties make PD-L1 a promising target for cancer immunotherapy. Theclinical trials of anti-PD-L1 immunotherapy described in the Examplesdemonstrate for the first time that mAb blockade of the immuneinhibitory ligand, PD-L1, produces both durable tumor regression andprolonged (≥24 weeks) disease stabilization in patients with metastaticNSCLC, MEL, RCC and OV, including those with extensive prior therapy.The human anti-PD-L1 HuMAb, BMS-936559, had a favorable safety profileoverall at doses up to and including 10 mg/kg, as is evident from thelow (9%) incidence of grade 3-4 drug-related AEs. These findings areconsistent with the mild autoimmune phenotype seen in PD-L1^(−/−) mice(Dong et al., 2004) and the more severe hyperproliferation seen inCTLA-4^(−/−) mice relative to PD-1^(−/−) mice (Phan et al., 2003; Tivolet al., 1995; Nishimura et al., 1999). Most of the toxicities associatedwith anti-PD-L1 administration in patients were immune-related,suggesting on-target effects. The spectrum and frequency of adverseevents of special interest (AEOSIs) is somewhat different betweenanti-PD-L1 and anti-CTLA-4, emphasizing the distinct biology of thesepathways (Ribas et al., 2005). Infusion reactions were observed withBMS-936559, although they were mild in most patients. Severe colitis, adrug-related AE observed in ipilimumab-treated patients (Beck et al.,2006), was infrequently noted with anti-PD-L1.

As noted above for anti-PD-1 immunotherapy, another important feature ofanti-PD-L1 therapy is the durability of responses across multiple tumortypes. This is particularly notable considering the advanced disease andprior treatment of patients on the current study. Although not compareddirectly, this durability appears greater than that observed with mostchemotherapies and kinase inhibitors used in these diseases.

Because peripheral blood T-cells express PD-L1, it is possible to assessin vivo RO by BMS-963559 as a pharmacodynamic measure. Median RO was65.8%, 66.2%, and 72.4% for the doses tested. Whereas these studiesprovide a direct assessment and evidence of target engagement inpatients treated with BMS-936559, relationships between RO in peripheralblood and the tumor microenvironment remain poorly understood.

Based on the clinical data disclosed herein, this disclosure provides amethod for immunotherapy of a subject afflicted with cancer, whichmethod comprises administering to the subject a composition comprising atherapeutically effective amount of an anti-PD-L1 Ab of the invention oran antigen-binding portion thereof. The disclosure also provides amethod of inhibiting growth of tumor cells in a subject, comprisingadministering to the subject an anti-PD-L1 Ab of the invention or anantigen-binding portion thereof. In preferred embodiments, the subjectis a human. In certain embodiments, the Ab or antigen-binding portionthereof is of an IgG1 or IgG4 isotype. In certain embodiments, the Ab orantigen-binding portion thereof is a mAb or an antigen-binding portionthereof. In certain other embodiments, the Ab or antigen-binding portionthereof is a chimeric, humanized or human Ab or an antigen-bindingportion thereof. In preferred embodiments for treating human subjects,the Ab or antigen-binding portion thereof is a human Ab or anantigen-binding portion thereof.

Clinical trials described in the Examples employed the anti-PD-L1 HuMAbBMS-936559 to treat cancer. While BMS-936559 (designated HuMAb 12A4 inU.S. Pat. No. 7,943,743) was selected as the lead anti-PD-L1 Ab forentering the clinic, it is notable that several anti-PD-L1 Abs of theinvention share with 12A4 functional properties that are important tothe therapeutic activity of 12A4, including high affinity bindingspecifically to human PD-L1, increasing T-cell proliferation, IL-2secretion and interferon-γ production in an MLR assay, inhibiting thebinding of PD-L1 to PD-1, and reversing the suppressive effect of Tregulatory cells on T cell effector cells and/or dendritic cells.Moreover, certain of the anti-PD-L1 Abs of the invention, namely 1B12,7H1 and 12B7 are structurally related to 12A4 in comprising V_(H) andV_(κ) regions that have sequences derived from V_(H) 1-69 and V_(κ) L6germline sequences, respectively. In addition, at least 12B7, 3G10, 1B12and 13G4 cross-compete with 12A4 for binding to the same epitope regionof hPD-L1, whereas 5F8 and 10A5 may bind to the same or an overlappingepitope region as 12A4 (Examples 2 and 3). Thus, the preclinicalcharacterization of 12A4 and other anti-PD-L1 HuMabs indicate that themethods of treating cancer provided herein may be performed using any ofthe broad genus of anti-PD-L1 Abs of the invention.

Accordingly, this disclosure provides immunotherapy methods comprisingadministering to a patient an anti-PD-L1 Ab or antigen-binding portionthereof comprising (a) a heavy chain variable region that comprisesconsecutively linked amino acids having a sequence derived from a humanV_(H) 1-18 germline sequence, and a light chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(κ) L6 germline sequence; (b) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 1-69 germline sequence, and a light chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(κ) L6 germline sequence; (c) a heavychain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(H) 1-3 germline sequence, anda light chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L15 germlinesequence; (d) a heavy chain variable region that comprises consecutivelylinked amino acids having a sequence derived from a human V_(H) 1-69germline sequence, and a light chain variable region that comprisesconsecutively linked amino acids having a sequence derived from a humanV_(κ) A27 germline sequence; (e) a heavy chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(H) 3-9 germline sequence, and a light chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(κ) L15 germline sequence; or (f) a heavy chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(H) 3-9 germline sequence, and a lightchain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(κ) L18 germline sequence.

In certain other embodiments, the anti-PD-L1 Ab or antigen-bindingportion thereof administered to the patient cross-competes for bindingto PD-L1 with a reference Ab or a reference antigen-binding portionthereof comprising: (a) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 15 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 25; (b) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 16 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 26; (c) a human heavy chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 17 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 27; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 18 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 28; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 19 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 29; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 20 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 30; (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 21 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEO ID NO: 31; (h) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 22 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 32; (i) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 23 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEO ID NO: 33; or (j) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 24 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 34. In preferred embodiments, the Ab orantigen-binding portion thereof cross-competes for binding to PD-1 witha reference Ab or reference antigen-binding portion thereof comprising ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 16 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 26.

In certain preferred embodiments of the immunotherapy methods disclosedherein, the anti-PD-L1 Ab or antigen-binding portion thereofadministered to the subject comprises: (a) a human heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 15 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 25; (b) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 16 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 26; (c) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 17 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 27; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 18 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 28; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 19 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 29; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 20 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 30; (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 21 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEO ID NO: 31; (h) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 22 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 32; (i) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 23 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEO ID NO: 33; or (j) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 24 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 34. In more preferred embodiments, the anti-PD-L1 Abor antigen-binding portion comprises a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 16 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 26.

Broad Spectrum of Cancers Treatable by Anti-PD-L1 Immunotherapy

The clinical activity of anti-PD-L1 in patients with advanced NSCLC,similar to the activity of anti-PD-1 in these patients, was surprisingand unexpected since NSCLC has been considered to be poorly responsiveto immune-based therapies (Holt and Disis, 2008; Holt et al., 2011). Thepresent clinical data obtained with BMS-936559, an anti-PD-L1 Ab of theinvention, substantiate and extend the evidence obtained using theanti-PD-1 Ab that immunotherapy based on PD-1 blockade is not applicableonly to “immunogenic” tumor types, such as MEL and RCC, but is alsoeffective with a broad range of cancers, including treatment-refractorymetastatic NSCLC, that are generally not considered to beimmune-responsive. Preferred cancers that may be treated using theanti-PD-L1 Abs of the invention include MEL (e.g., metastatic malignantmelanoma), RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovarian cancer(OV), gastric cancer (GC), breast cancer (BC), pancreatic carcinoma (PC)and carcinoma of the esophagus. Additionally, the invention includesrefractory or recurrent malignancies whose growth may be inhibited usingthe anti-PD-L1 Abs of the invention.

Examples of other cancers that may be treated using an anti-PD-L1 Ab inthe methods of the invention, based on the indications of very broadapplicability of anti-PD-L1 immunotherapy provided herein, include bonecancer, skin cancer, cancer of the head or neck, breast cancer, lungcancer, cutaneous or intraocular malignant melanoma, renal cancer,uterine cancer, castration-resistant prostate cancer, colon cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, carcinomas of the ovary, gastrointestinal tractand breast, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma, multiplemyeloma, environmentally induced cancers including those induced byasbestos, metastatic cancers, and any combinations of said cancers. Thepresent invention is also applicable to treatment of metastatic cancers.

Combination Therapy with Anti-PD-L1 Abs

Optionally, Abs to PD-L1 can be combined with an immunogenic agent, forexample a preparation of cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),antigen-presenting cells such as dendritic cells bearingtumor-associated antigens, and cells transfected with genes encodingimmune stimulating cytokines (He et al., 2004). Non-limiting examples oftumor vaccines that can be used include peptides of melanoma antigens,such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF.PD-1 blockade may also be effectively combined with standard cancertreatments, including chemotherapeutic regimes, radiation, surgery,hormone deprivation and angiogenesis inhibitors, as well as anotherimmunotherapeutic Ab (e.g., an anti-PD-1, anti-CTLA-4 or anti-LAG-3 Ab).

Uses of Anti-PD-L1 Abs

This disclosure provides the use of any anti-PD-L1 Ab or antigen-bindingportion thereof of the invention for the preparation of a medicament forinhibiting signaling from the PD-1/PD-L1 pathway so as to therebypotentiate an endogenous immune response in a subject afflicted withcancer. The disclosure also provides the use of any anti-PD-L1 Ab orantigen-binding portion thereof of the invention for the preparation ofa medicament for immunotherapy of a subject afflicted with cancercomprising disrupting the interaction between PD-1 and PD-L1. Thedisclosure provides medical uses of any anti-PD-L1 Ab or antigen-bindingportion thereof of the invention corresponding to all the embodiments ofthe methods of treatment employing an anti-PD-L1 Ab described herein.

This disclosure also provides an anti-PD-L1 Ab or an antigen-bindingportion thereof of the invention for use in potentiating an endogenousimmune response in a subject afflicted with cancer by inhibitingsignaling from the PD-1/PD-L1 pathway. The disclosure further providesan anti-PD-L1 Ab or an antigen-binding portion thereof of the inventionfor use in immunotherapy of a subject afflicted with cancer comprisingdisrupting the interaction between PD-1 and PD-L1. These Abs may be usedin potentiating an endogenous immune response against, or inimmunotherapy of, the full range of cancers disclosed herein. Inpreferred embodiments, the cancers include MEL (e.g., metastaticmalignant MEL), RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovariancancer (OV), gastric cancer (GC), breast cancer (BC), pancreaticcarcinoma (PC) and carcinoma of the esophagus.

Validation of Cancer Immunotherapy by Immune Checkpoint Blockade

A major implication of the clinical activity of immune checkpointblockade is that significant endogenous immune responses to tumorantigens are generated and these responses may be harnessedtherapeutically to mediate clinical tumor regression upon checkpointinhibition. In fact, there is evidence that inhibitory ligands such asPD-L1 are induced in response to immune attack, a mechanism termedadaptive resistance (Gajewski et al., 2010; Taube et al., 2012). Thispotential mechanism of immune resistance by tumors suggests thatPD-1/PD-L1-directed therapy might synergize with other treatments thatenhance endogenous antitumor immunity. Follow-up studies have verifiedthat patients continue to demonstrate tumor control after cessation ofPD-1/PD-L1 pathway blockade (Lipson et al., 2013). Such tumor controlmay reflect a persistent antitumor immune response and the generation ofeffective immunologic memory to enable sustained control of tumorgrowth.

The data disclosed herein on the clinical testing of Abs that block theimmunoregulatory receptor, PD-1, and also of Abs that block one of itscognate ligands, PD-L1, are unprecedented. These data constitute thelargest clinical experience to date with PD-1 pathway-directed cancerimmunotherapy, and the first report specifically describing the safety,tolerability, and initial clinical activity of an anti-PD-L1-directedagent. These findings show that both anti-PD-1 and anti-PD-L1 havefavorable overall safety profiles and provide clear evidence of clinicalactivity across diverse cancers, including NSCLC, a tumor nothistorically considered responsive to immunotherapy, as well as tumorsknown to respond to immunotherapy, including MEL, RCC and OV. Thus,these data strongly validate the PD-1/PD-L1 pathway as an importanttarget for therapeutic intervention in cancer.

The remarkable similarities observed between the patterns of clinicalactivity obtained with the anti-PD-1 and anti-PD-L1 mAbs, and among thetumor types analyzed to date, validate the general importance of thePD-1/PD-L1 signaling pathway in tumor immune resistance and as a targetfor therapeutic intervention. Although the molecular interactionsblocked by these two Abs are not identical, it has been clearlydemonstrated herein that, irrespective of mechanistic details, bothanti-PD-1 and anti-PD-L1 Abs of the invention are effective in treatingpatients afflicted with a wide variety of cancers, including“immunogenic” cancers such as MEL and RCC as well astreatment-refractory metastatic NSCLC, a tumor that is generally notconsidered to be immune-responsive. In certain embodiments of theinvention, either or both of these Abs can be administered incombination with another therapeutic agent such as a cytokine.

Infectious Diseases

Other methods of the invention are used to treat patients that have beenexposed to particular toxins or pathogens. For example, another aspectof the disclosure provides a method of treating an infectious disease ina subject comprising administering to the subject an anti-PD1 or ananti-PD-L1 Ab, or antigen-binding portion thereof, of the invention suchthat the subject is treated for the infectious disease. Preferably, theAb is a human anti-human PD-1 or PD-L1 Ab (such as any of the human Absdescribed herein). Alternatively, the Ab is a chimeric or humanized Ab.

Similar to its application to tumors as discussed above, Ab-mediatedPD-1 or PD-L1 blockade can be used alone, or as an adjuvant, incombination with vaccines, to potentiate an immune response topathogens, toxins, and/or self-antigens. Examples of pathogens for whichthis therapeutic approach may be particularly useful include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, and C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonasaeruginosa. PD-1 and/or PD-L1 blockade is particularly useful againstestablished infections by agents such as HIV that present alteredantigens over the course of an infection. Novel epitopes on theseantigens are recognized as foreign at the time of anti-human PD-1 orPD-L1 administration, thus provoking a strong T cell response that isnot dampened by negative signals through the PD-1/PD-L1 pathway.

In the above methods, PD-1 or PD-L1 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., administrationof interferons, GM-CSF, G-CSF or IL-2).

Kits

Also within the scope of the present invention are kits, includingpharmaceutical kits, comprising an anti-PD-1 and/or an anti-PD-L1 Ab ofthe invention for therapeutic uses, and diagnostic kits comprising ananti-PD-L1 Ab of the invention for assaying membranous PD-L1 expressionas a biomarker for screening patients for immunotherapy or forpredicting the efficacy of an immunotherapeutic agent. Kits typicallyinclude a label indicating the intended use of the contents of the kitand instructions for use. The term label includes any writing, orrecorded material supplied on or with the kit, or which otherwiseaccompanies the kit. In certain embodiments of a pharmaceutical kit, theanti-PD-1 and/or anti-PD-L1 Abs may be co-packaged with othertherapeutic agents in unit dosage form. In certain embodiments of adiagnostic kit, the anti-PD-L1 Ab may be co-packaged with other reagentsfor performing an assay to detect and/or quantify PD-L1 expression.

In certain preferred embodiments, the pharmaceutical kit comprises theanti-human PD-1 HuMAb, nivolumab. In other preferred embodiments, thepharmaceutical kit comprises the anti-human PD-L1 HuMAb, BMS-936559. Incertain preferred embodiments, the diagnostic kit comprises the rabbitanti-human PD-L1 mAb, 28-8, comprising the V_(H) and V_(κ) regions whoseamino acid sequences are set forth in SEQ ID NOs. 35 and 36,respectively. In other preferred embodiments, the diagnostic kitcomprises the murine anti-human PD-L1 mAb, 5H1 (Dong et al., 2002).

PD-L1 Biomarker for Predicting Anti-PD-1 Efficacy

A particular challenge in cancer immunotherapy has been theidentification of mechanism-based predictive biomarkers to enablepatient selection and guide on-treatment management. Data disclosed inthe Examples below indicate that cell surface PD-L1 expression in tumorsis a useful molecular marker for predicting the efficacy of, andselecting patients for, anti-PD-1 immunotherapy.

There are conflicting reports in the literature about the clinicalimplications of PD-L1 being expressed in tumors. Several studies haveconcluded that PD-L1 expression in tumors correlates with a poorprognosis for the patient. See, e.g., Hino et al., 2010 (MEL); Hamanishiet al., 2007 (OV); Thompson et al., 2006 (RCC). These findings may berationalized on the basis that the interaction of PD-L1 on tumor cellsand PD-1 on T cells helps abrogate immune responses directed against thetumor, resulting in immune evasion from tumor-specific T cells. However,in contrast to the foregoing studies, Gadiot et al., 2011 and Taube etal., 2012 have recently reported that PD-L1 expression in melanomatumors correlates with a trend toward better survival. These seeminglycontradictory data may reflect the relatively small numbers of patientsanalyzed, different histologic subtypes studied, or differentmethodologies used, e.g., the use of different Abs to stain PD-L1, theuse of frozen versus paraffin-embedded material for IHC, and thedetection of membranous and/or cytoplasmic staining of PD-L1. Taube etal., 2012 note that PD-L1 is a type I transmembrane molecule, andhypothesize that while the cytoplasmic presence of PD-L1 may representintracellular stores of this polypeptide that may be deployed to thecell surface upon appropriate stimulation, it is cell surface PD-L1expression of that is biologically relevant as a potential biomarker forpredicting clinical response to PD-1 blockade. See, also, Brahmer etal., 2010, which describes preliminary evidence, obtained on a smallsample size of only 9 patients, of a correlation between membranousPD-L1 expression and anti-PD-1 efficacy. The data described in theExamples below on the use of membranous PD-L1 expression as a biomarkerfor anti-PD-1 efficacy, which was obtained from analysis of a muchlarger sample, substantiate the hypothesis that PD-L1 expression may beused as a biomarker for predicting anti-PD-1 clinical response and forscreening patients to identify suitable candidates for immunotherapywith an anti-PD-1 Ab or other inhibitors of inhibitory immunoregulators.

Specifically, membranous PD-L1 expression was assayed using an automatedIHC protocol and a rabbit anti-hPD-L1 Ab. Strikingly, in the initial setof data analyzed (see Example 8), no patients with cell surfacePD-L1-negative tumors (MEL, NSCLC, CRC, RCC and CRPC) experienced an ORfollowing treatment with the anti-PD-1 Ab, nivolumab. In contrast, cellsurface expression of PD-L1 on tumor cells in pretreatment biopsies maybe associated with an increased rate of OR among patients treated withnivolumab. While tumor cell expression of PD-L1 may be driven byconstitutive oncogenic pathways, it may also reflect “adaptive immuneresistance” in response to an endogenous antitumor immune response, partof a host inflammatory response, which may remain in check unlessunleashed by blockade of the PD-1/PD-L1 pathway (Taube et al., 2012).This emerging concept of adaptive immune resistance in cancer immunologysuggests that inhibitory ligands such as PD-L1 are induced in responseto immune attack (Gajewski et al., 2010; Taube et al., 2012). A majorimplication of the clinical activity of immune checkpoint blockade asdescribed herein is that significant endogenous immune responses totumor antigens are generated and these responses may be harnessedtherapeutically to mediate clinical tumor regression upon checkpointinhibition. This potential mechanism of immune resistance by tumorssuggests that PD-1/PD-L1-directed therapy might synergize with othertreatments that enhance endogenous antitumor immunity. It also suggeststhat cell surface expression of PD-L1 in tumors and/or inflammatorycells in the tumor microenvironment may be a marker not just fortreatment of cancer patients with an anti-PD-1 Ab, but also fortreatment with an anti-PD-L1 Ab as well as treatments targetinginhibitory immunoregulatory pathways other than the PD-1/PD-L1 pathway.

Assaying Cell-Surface PD-L1 Expression by Automated IHC

As described in the Examples, an automated IHC method was developed forassaying the expression of PD-L1 on the surface of cells in FFPE tissuespecimens. The disclosure provides methods for detecting the presence ofhuman PD-L1 antigen in a test tissue sample, or quantifying the level ofhuman PD-L1 antigen or the proportion of cells in the sample thatexpress the antigen, which methods comprise contacting the test sample,and a negative control sample, with a mAb that specifically binds tohuman PD-L1, under conditions that allow for formation of a complexbetween the Ab or portion thereof and human PD-L1. Preferably, the testand control tissue samples are FFPE samples. The formation of a complexis then detected, wherein a difference in complex formation between thetest sample and the negative control sample is indicative of thepresence of human PD-L1 antigen in the sample. Various methods are usedto quantify PD-L1 expression.

In a particular embodiment, the automated IHC method comprises: (a)deparaffinizing and rehydrating mounted tissue sections in anautostainer; (b) retrieving antigen using a decloaking chamber and pH 6buffer, heated to 110° C. for 10 min; (c) setting up reagents on anautostainer; and (d) running the autostainer to include steps ofneutralizing endogenous peroxidase in the tissue specimen; blockingnon-specific protein-binding sites on the slides; incubating the slideswith primary Ab; incubating with a post-primary blocking agent;incubating with NovoLink Polymer; adding a chromogen substrate anddeveloping; and counterstaining with hematoxylin.

For assessing PD-L1 expression in tumor tissue samples, a pathologistexamines the number of membrane PD-L1⁺ tumor cells in each field under amicroscope and mentally estimates the percentage of cells that arepositive, then averages them to come to the final percentage. Thedifferent staining intensities are defined as 0/negative, 1+/weak,2+/moderate, and 3+/strong. Typically, percentage values are firstassigned to the 0 and 3+ buckets, and then the intermediate 1+ and 2+intensities are considered. For highly heterogeneous tissues, thespecimen is divided into zones, and each zone is scored separately andthen combined into a single set of percentage values. The percentages ofnegative and positive cells for the different staining intensities aredetermined from each area and a median value is given to each zone. Afinal percentage value is given to the tissue for each stainingintensity category: negative, 1+, 2+, and 3+. The sum of all stainingintensities needs to be 100%.

Staining is also assessed in tumor-infiltrating inflammatory cells suchas macrophages and lymphocytes. In most cases macrophages serve as aninternal positive control since staining is observed in a largeproportion of macrophages. While not required to stain with 3+intensity, an absence of staining of macrophages should be taken intoaccount to rule out any technical failure. Macrophages and lymphocytesare assessed for plasma membrane staining and only recorded for allsamples as being positive or negative for each cell category. Stainingis also characterized according to an outside/inside tumor immune celldesignation. “Inside” means the immune cell is within the tumor tissueand/or on the boundaries of the tumor region without being physicallyintercalated among the tumor cells. “Outside” means that there is nophysical association with the tumor, the immune cells being found in theperiphery associated with connective or any associated adjacent tissue.

In certain embodiments of these scoring methods, the samples are scoredby two pathologists operating independently and the scores aresubsequently consolidated. In certain other embodiments, theidentification of positive and negative cells is scored usingappropriate software.

A histoscore is used as a more quantitative measure of the IHC data. Thehistoscore is calculated as follows:

Histoscore=[(% tumor×1(low intensity))+(% tumor×2(medium intensity))+(%tumor×3(high intensity)]

To determine the histoscore, the pathologist estimates the percentage ofstained cells in each intensity category within a specimen. Becauseexpression of most biomarkers is heterogeneous the histoscore is a truerrepresentation of the overall expression. The final histoscore range is0 (no expression) to 300 (maximum expression).

An alternative means of quantifying PD-L1 expression in a test tissuesample IHC is to determine the adjusted inflammation score (AIS) scoredefined as the density of inflammation multiplied by the percent PD-L1expression by tumor-infiltrating inflammatory cells (Taube et al.,2012).

Cancer Immunotherapy with Anti-PD-1 Comprising a Patient Selection Step

This disclosure also provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises: (a) selecting a subjectthat is a suitable candidate for immunotherapy, the selecting comprising(i) optionally providing a test tissue sample obtained from a patientwith cancer of the tissue, the test tissue sample comprising tumor cellsand tumor-infiltrating inflammatory cells, (ii) assessing the proportionof cells in the test tissue sample that express PD-L1 on the cellsurface, and (iii) selecting the subject as a suitable candidate basedon an assessment that the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface exceeds a predetermined thresholdlevel; and (b) administering to the selected subject a compositioncomprising a therapeutically effective amount of an agent that inhibitssignaling from an inhibitory immunoregulator.

There is evidence that membranous PD-L1 expression is a surrogate for anendogenous antitumor immune response that is part of a host inflammatoryresponse (Gajewski et al., 2010; Taube et al., 2012). Accordingly, cellsurface expression of PD-L1 in tumors and/or tumor-infiltratinginflammatory cells is a marker not just for selecting cancer patientswho would benefit from treatment with an anti-PD-1 Ab, but also fortreatment with an anti-PD-L1 Ab as well as treatments targetinginhibitory immunoregulatory pathways other than the PD-1/PD-L1 pathway.For example, cell surface expression of PD-L1 in tumors and/ortumor-infiltrating inflammatory cells may be used as a marker foridentifying or selecting suitable cancer patients who would benefit fromimmunotherapy with agents, including Abs, that target, and disrupt orinhibit signaling from, inhibitory immunoregulators such as PD-L1,Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), B and T Lymphocyte Attenuator(BTLA), T cell Immunoglobulin and Mucin domain-3 (TIM-3), LymphocyteActivation Gene-3 (LAG-3), Killer cell Lectin-like Receptor G1 (KLRG-1),Natural Killer Cell Receptor 2B4 (CD244), and CD160 (Baitsch et al.,2012). In certain preferred embodiments, the inhibitory immunoregulatoris a component of the PD-1/PD-L1 signaling pathway. In other preferredembodiments, the inhibitory immunoregulator is an anti-PD-1 Ab of theinvention. In yet other preferred embodiments, the inhibitoryimmunoregulator is an anti-PD-L1 Ab of the invention.

Although many of the immunotherapy methods below comprising assayingPD-L1 expression, i.e., employing a PD-L1 expression biomarker, aredescribed as comprising the selection of a patient that is, or is not,suitable for anti-PD-1 immunotherapy or as comprising the administrationof an anti-PD-1 Ab for immunotherapeutic purposes, it should beunderstood that these methods apply generally to the selection of apatient that is, or is not, suitable for immunotherapy with, or to theadministration of an inhibitor of an inhibitory immunoregulator or acomponent or ligand thereof. Further, in any the methods comprising themeasurement of PD-L1 expression in a test tissue sample, it should beunderstood that the step comprising the provision of a test tissuesample obtained from a patient is an optional step. That is, in certainembodiments the method includes this step, and in other embodiments,this step is not included in the method. It should also be understoodthat in certain preferred embodiments the “assessing” step to identify,or determine the number or proportion of, cells in the test tissuesample that express PD-L1 on the cell surface is performed by atransformative method of assaying for PD-L1 expression, for example byperforming a reverse transcriptase-polymerase chain reaction (RT-PCR)assay or an IHC assay. In certain other embodiments, no transformativestep is involved and PD-L1 expression is assessed by, for example,reviewing a report of test results from a laboratory. In certainembodiments, the steps of the methods up to, and including, assessingPD-L1 expression provides an intermediate result that may be provided toa physician or other medical practitioner for use in selecting asuitable candidate for immunotherapy and/or administering animmunotherapeutic agent to the patient. In certain embodiments, thesteps that provide the intermediate result may be performed by a medicalpractitioner or someone acting under the direction of a medicalpractitioner. In other embodiment, these steps are performed by anindependent person or laboratory.

The disclosure further provides a method for treatment of a subjectafflicted with cancer, which method comprises: (a) selecting a subjectthat is not suitable for treatment with an agent that inhibits aninhibitory immunoregulator, e.g., anti-PD-1 Ab immunotherapy, theselecting comprising (i) optionally providing a test tissue sampleobtained from a patient with cancer of the tissue, the test tissuesample comprising tumor cells and tumor-infiltrating inflammatory cells;(ii) assessing the proportion of cells in the test tissue sample thatexpress PD-L1 on the surface of the cells; and (iii) selecting thesubject as not suitable for immunotherapy with an inhibitor of aninhibitory immunoregulator, e.g., an anti-PD-1 Ab, based on anassessment that the proportion of cells in the test tissue sample thatexpress PD-L1 on the cell surface is less than a predetermined thresholdlevel; and (b) administering a standard-of-care therapeutic other thanan inhibitor of an inhibitory immunoregulator, e.g., an anti-PD-1 Ab, tothe selected subject.

Measurement of PD-L1 Expression

In certain embodiments of any of the present methods, the proportion ofcells that express PD-L1 is assessed by performing an assay to determinethe presence of PD-L1 RNA. In further embodiments, the presence of PD-L1RNA is determined by RT-PCR, in situ hybridization or RNase protection.In other embodiments, the proportion of cells that express PD-L1 isassessed by performing an assay to determine the presence of PD-L1polypeptide. In further embodiments, the presence of PD-L1 polypeptideis determined by immunohistochemistry (IHC), enzyme-linked immunosorbentassay (ELISA), in vivo imaging, or flow cytometry. In preferredembodiments, PD-L1 expression is assayed by IHC. Flow cytometry may beparticularly suitable for assaying PD-L1 expression in cells ofhematologic tumors. In preferred embodiments of all of these methods,cell surface expression of PD-L1 is assayed using, e.g., IHC or in vivoimaging.

Imaging techniques have provided important tools in cancer research andtreatment. Recent developments in molecular imaging systems, includingpositron emission tomography (PET), single-photon emission computedtomography (SPECT), fluorescence reflectance imaging (FRI),fluorescence-mediated tomography (FMT), bioluminescence imaging (BLI),laser-scanning confocal microscopy (LSCM) and multiphoton microscopy(MPM), will likely herald even greater use of these techniques in cancerresearch. Some of these molecular imaging systems allow clinicians tonot only see where a tumor is located in the body, but also to visualizethe expression and activity of specific molecules, cells, and biologicalprocesses that influence tumor behavior and/or responsiveness totherapeutic drugs (Condeelis et al., 2010). Ab specificity, coupled withthe sensitivity and resolution of PET, makes immunoPET imagingparticularly attractive for monitoring and assaying expression ofantigens in tissue samples (McCabe et al., 2010; Olafsen et al., 2010).In certain embodiments of any of the present methods, PD-L1 expressionis assayed by immunoPET imaging.

In certain embodiments of any of the present methods, the proportion ofcells in a test tissue sample that express PD-L1 is assessed byperforming an assay to determine the presence of PD-L1 polypeptide onthe surface of cells in the test tissue sample. In certain embodiments,the test tissue sample is a FFPE tissue sample. In certain preferredembodiments, the presence of PD-L1 polypeptide is determined by IHCassay. In further embodiments, the IHC assay is performed using anautomated process. In further embodiments, the IHC assay is performedusing an anti-PD-L1 mAb to bind to the PD-L1 polypeptide.

Abs that Bind Specifically to Cell-Surface-Expressed PD-L1 in FFPETissues

An Ab may bind to an antigen in fresh tissues but completely fail torecognize the antigen in an FFPE tissue sample. This phenomenon, wellknown in the art, is thought to be due primarily to intra- andinter-molecular cross-linking of polypeptides induced by formalinfixation, which alters the epitope recognized by the Ab (Sompuram etal., 2006). In addition, several factors known to influence staining inFFPE tissue, including variable time to fixation, inadequate fixationperiod, differences in fixative used, tissue processing, Ab clone anddilution, antigen retrieval, detection system, and interpretation ofresults using different threshold points are important variables thatcan affect tissue antigenicity and IHC measurements (Bordeaux et al.,2010). In particular, a lack of anti-human PD-L1 Abs that stain PD-L1 inFFPE specimens has been noted in the art (Hamanishi et al., 2007). Thus,the contradictory results reported by different groups on theimplications of PD-L1 expression for prognosis of a tumor may, in part,reflect the differential abilities of anti-PD-L1 Abs used to detectPD-L1 polypeptide in FFPE tissue samples. Indeed, our analysis of fivecommercially available anti-hPD-L1 Abs shows that these Abs failed todistinguish FFPE cells expressing PD-L1 from cells that did not expressPD-L1 (see Example 9, Table 6). Accordingly, in order to detect hPD-L1on the surface of cells using an IHC assay on FFPE tissues, there is aneed for anti-hPD-L1 Abs that bind specifically to cellsurface-expressed PD-L1 in FFPE tissue samples.

This disclosure provides a mAb or an antigen-binding portion thereofthat binds specifically to a cell surface-expressed PD-L1 antigen in aFFPE tissue sample. In preferred embodiments, the mAb or antigen-bindingportion thereof does not bind to a cytoplasmic PD-L1 polypeptide in theFFPE tissue sample or exhibits a very low level of background binding.In certain other embodiments, the presence or absence of bindingspecifically to a cell surface-expressed or a cytoplasmic PD-L1polypeptide is detected by immunohistochemical staining. In certainpreferred aspects of the invention, the mAb or antigen-binding portionis a rabbit Ab or a portion thereof. In other preferred embodiments, themAb is the rabbit mAb designated 28-8, 28-1, 28-12, 29-8 or 20-12. Inmore preferred embodiments, the mAb is the rabbit mAb designated 28-8 oran antigen-binding portion thereof. In further embodiments, the mAb isan Ab comprising a heavy chain variable region (V_(H)) comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 35 and a light chain variable region (V_(κ)) comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 36. In other embodiments, the mAb comprises the CDR1, CDR2 and CDR3domains in a V_(H) having the sequence set forth in SEQ ID NO: 35, andthe CDR1, CDR2 and CDR3 domains in a V_(κ) having the sequence set forthin SEQ ID NO: 36.

It is known in the art that rabbit Abs have certain advantages overmurine Abs. For example, rabbit Abs generally exhibit more diverseepitope recognition, improved immune response to small-size epitopes,and higher specificity and affinity compared to murine Abs (see, e.g.,Fischer et al., 2008; Cheang et al., 2006; Rossi et al., 2005). Forexample, the rabbit's lower immune dominance and larger B-cellrepertoire results in greater epitope recognition compared to murineAbs. Further, the high specificity and novel epitope recognition ofrabbit antibodies translates to success with recognition ofpost-translational modifications (Epitomics, 2013). In addition, manyprotein targets relevant to signal transduction and disease are highlyconserved between mice, rats and humans, and can therefore be recognizedas self-antigens by a mouse or rat host, making them less immunogenic.This problem is avoided by generating Abs in rabbits. In addition, inapplications in which two antigen-specific Abs are required, it is moreconvenient to have the Abs come from two different species. Thus, forexample, it is easier to multiplex a rabbit Ab such as 28-8 with otherAbs (likely to be murine Abs since the best immune marking Abs aremurine Abs) that can mark immune cells that also express PD-L1 (e.g.,macrophages and lymphocytes). Thus, rabbit anti-hPD-L1 mAbs, e.g., 28-8,are particularly suited to IHC assays for detecting surface-expressedPD-L1 in FFPE tissue samples and have distinct advantages over murineAbs, such as 5H1.

As described in Example 9, a large number (185) of antibody multiclonesfrom both rabbit and mouse immunizations were screened, and only tenrabbit Ab, but no mouse Ab, multiclones specifically detected themembranous form of hPD-L1. After further extensive screening by multiplerounds of IHC, 15 purified rabbit subclones were selected based on theirspecificity and intensity of staining (see Table 4). Following furthercharacterization of the antibodies to determine their binding affinityand cross-competition by surface plasmon resonance, as well as screeningby IHC on FFPE tissues, mAb 28-8 was selected as the Ab with the bestcombination of binding to membranous PDF-L1 with high affinity andspecificity, and low background staining.

In certain aspects of this invention, the mAb or antigen-binding portioncross-competes with mouse mAb 5H1 for binding to PD-L1, which indicatesthat these antibodies bind to the same epitope region of PD-L1. Incertain other aspects, the mAb or antigen-binding portion thereof doesnot cross-compete with mouse mAb 5H1 for binding to PD-L1, indicatingthat they do not bind to the same epitope region of PD-L1.

The disclosure also provides nucleic acids encoding all of the rabbitanti-hPD-L1 Abs or portions thereof disclosed herein.

Immunotherapeutic Methods Comprising Measurement of Cell Surface PD-L1Expression

The availability of rabbit Abs that bind with high affinity specificallyto membranous PD-L1 in FFPE tissue specimens facilitates methodscomprising a step of detecting PD-L1 polypeptide on the surface of cellsin FFPE tissue samples. Accordingly, this disclosure also provides amethod for immunotherapy of a subject afflicted with cancer, whichmethod comprises: (a) selecting a subject that is a suitable candidatefor immunotherapy, the selecting comprising: (i) optionally providing aFFPE test tissue sample obtained from a patient with cancer of thetissue, the test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells; (ii) assessing the proportion ofcells in the test tissue sample that express PD-L1 on the cell surfaceby IHC using a rabbit anti-human PD-L1 Ab, e.g., mAb 28-8, to bind tothe PD-L1; and (iii) selecting the subject as a suitable candidate basedon an assessment that the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface exceeds a predetermined thresholdlevel; and (b) administering a composition comprising a therapeuticallyeffective amount of an anti-PD-1 Ab to the selected subject.

In certain embodiments of methods employing IHC to assay PD-L1expression in FFPE tissues, an automated IHC assay is used. Theautomated IHC process is performed on an autostainer and comprises: (a)de-paraffinizing the FFPE sample with xylene and rehydrating the sample;(b) retrieving the antigen using a decloaking chamber; (c) blockingnonspecifc protein binding sites by incubation with a Protein Block; (d)incubating the sample with a primary anti-PD-L1 Ab; (e) adding apolymeric horseradish peroxidase (HRP)-conjugated secondary Ab; (f)detecting the bound secondary Ab comprising staining with a3,3′-diaminobenzidine (DAB) chromogen; and/or (g) counterstaining withhematoxylin. This automated IHC process has been optimized byminimization of the number of steps, optimization of incubation times,and selection of primary Abs, blocking and detection reagents thatproduce strong specific staining with a low level of backgroundstaining. In preferred embodiments of this automated IHC assay, theprimary anti-PD-L1 Ab is rabbit mAb 28-8 or murine mAb 5H1. In certainembodiments of this invention, this IHC assay, and any other IHC assaydescribed herein to measure PD-L1 expression, may be used as part of amethod of immunotherapy. In other embodiments, any of the IHC methodsdescribed herein is used independently of any therapeutic processrequiring the administration of a therapeutic, i.e., solely as adiagnostic method to assay PD-L1 expression.

In certain embodiments any of the immunotherapy methods describedherein, the Ab administered to the selected subject is any anti-PD-1 oranti-PD-L1 Ab or antigen-binding portion thereof of the invention. Incertain preferred embodiments, the subject is a human. In otherpreferred embodiments, the Ab is a human Ab or antigen-binding portionthereof. In more preferred embodiments, the anti-PD-1 Ab is nivolumaband the anti-PD-L1 Ab is BMS-936559. In certain other embodiments, theanti-PD-1 Ab is an Ab or antigen-binding portion thereof thatcross-competes with nivolumab for binding to PD-1, and the anti-PD-L1 Abis an Ab or antigen-binding portion thereof that cross-competes withBMS-936559 for binding to PD-L1. In certain preferred embodiments, thecancer to be treated is selected from the group consisting MEL, RCC,squamous NSCLC, non-squamous NSCLC, CRC, castration-resistant prostatecancer CRPC, HCC, squamous cell carcinoma of the head and neck,carcinomas of the esophagus, ovary, gastrointestinal tract and breast,and a hematological malignancy.

In certain embodiments of the disclosed methods, the predeterminedthreshold is based on a proportion of (a) tumor cells, (b)tumor-infiltrating inflammatory cells, (c) particular tumor-infiltratinginflammatory cells, e.g., TILs or macrophages, or (d) a combination oftumor cells and tumor-infiltrating inflammatory cells, in a test tissuesample that expresses PD-L1 on the cell surface. In certain embodiments,the predetermined threshold is at least 0.001% of tumor cells expressingmembranous PD-L1 as determined by IHC. In other embodiments, thepredetermined threshold is at least 0.01%, preferably at least 0.1%,more preferably at least 1% of tumor cells expressing membranous PD-L1,as determined by IHC. In certain embodiments, the predeterminedthreshold is at least 5% of tumor cells expressing membranous PD-L1 asdetermined by IHC. In certain embodiments, the predetermined thresholdis at least 0.01%, at least 0.1%, at least 1%, or at least 5% of tumorcells expressing membranous PD-L1 as determined by IHC, and/or a singletumor-infiltrating inflammatory cell expressing membranous PD-L1 asdetermined by IHC. In certain other embodiments, the predeterminedthreshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5%of a tumor-infiltrating inflammatory cell expressing membranous PD-L1 asdetermined by IHC. In certain other embodiments, the predeterminedthreshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5%of a tumor-infiltrating lymphocyte expressing membranous PD-L1 asdetermined by IHC. In certain other embodiments, the predeterminedthreshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5%of a tumor-infiltrating macrophage expressing membranous PD-L1 asdetermined by IHC. In yet other embodiments, the predetermined thresholdis at least a single tumor cell or a single tumor-infiltratinginflammatory cell expressing membranous PD-L1 as determined by IHC.Preferably, PD-L1 expression is assayed by automated IHC using mAb 28-8or 5H1 as the primary Ab.

This disclosure also provides a method for treatment of a subjectafflicted with cancer, which method comprises: (a) screening a pluralityof subjects to identify a subject that is not a suitable candidate forimmunotherapy comprising the administration of an anti-PD-1 Ab to thesubject, the screening comprising: (i) optionally providing test tissuesamples from the plurality of subjects, the test tissue samplescomprising tumor cells and tumor-infiltrating inflammatory cells; (ii)assessing the proportion of cells in the test tissue samples thatexpress PD-L1 on the surface of the cells; and (iii) selecting thesubject as a candidate that is not suitable for anti-PD-1 Abimmunotherapy based on an assessment that the proportion of cells thatexpress PD-L1 on the surface of cells in the subject's test tissuesample is below a predetermined threshold level; and (b) administering astandard-of-care therapeutic other than an anti-PD-1 Ab to the selectedsubject.

This disclosure also provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises: (a) screening a pluralityof subjects to identify a subject that is a suitable candidate forimmunotherapy, the screening comprising: (i) optionally providing testtissue samples from the plurality of subjects, the test tissue samplescomprising tumor cells and tumor-infiltrating inflammatory cells; (ii)assessing the proportion of cells in the test tissue samples thatexpress PD-L1 on the surface of the cells; and (iii) selecting thesubject as a candidate that is suitable for anti-PD-1 Ab immunotherapybased on an assessment that the proportion of cells in the test tissuesample that express PD-L1 on the cell surface exceeds a predeterminedthreshold level; and (b) administering a composition comprising atherapeutically effective amount of an anti-PD-1 Ab to the selectedsubject.

This disclosure further provides a method for treatment of a subjectafflicted with cancer, which method comprises: (a) screening a pluralityof subjects to identify a subject that is a suitable candidate for thetreatment, the screening comprising: (i) optionally providing testtissue samples from the plurality of subjects, the test tissue samplescomprising tumor cells and tumor-infiltrating inflammatory cells; (ii)assessing the proportion of cells in the test tissue samples thatexpress PD-L1 on the surface of the cells, wherein the subject isidentified as a suitable candidate for anti-PD-1 Ab immunotherapy if theproportion of cells in the tissue sample that express PD-L1 on the cellsurface exceeds a predetermined threshold level, and the subject isidentified as a candidate that is not a suitable candidate for anti-PD-1Ab immunotherapy if the proportion of cells in the tissue sample thatexpress PD-L1 on the cell surface is below a predetermined thresholdlevel; and (b) administering a composition comprising a therapeuticallyeffective amount of an anti-PD-1 Ab to the subject identified as asuitable candidate for anti-PD-1 Ab immunotherapy, or (c) administeringa standard-of-care therapeutic other than an anti-PD-1 Ab to the subjectidentified as not a suitable candidate for anti-PD-1 Ab immunotherapy.

One aspect of this invention is a method for immunotherapy of a subjectafflicted with cancer, which method comprises: (a) optionally providinga test tissue sample obtained from a patient with cancer of the tissue,the test tissue sample comprising tumor cells and tumor-infiltratinginflammatory cells; (b) determining that a proportion of cells in thetest tissue sample express PD-L1 above a predetermined threshold levelon the cell surface; and (c) based on that determination administering acomposition comprising a therapeutically effective amount of ananti-PD-1 Ab to the subject. Another aspect of the invention is a methodfor treatment of a subject afflicted with cancer, which methodcomprises: (a) optionally providing a test tissue sample obtained from apatient with cancer of the tissue, the test tissue sample comprisingtumor cells and tumor-infiltrating inflammatory cells; (b) determiningthat the proportion of cells in the test tissue sample that expressPD-L1 on the cell surface is below a predetermined threshold level; and(c) based on that determination administering a standard-of-caretherapeutic other than an anti-PD-1 Ab to the subject.

Yet another aspect of the invention is a method for immunotherapy of asubject afflicted with cancer, which method comprises: (a) optionallyproviding a test tissue sample obtained from a patient with cancer ofthe tissue, the test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells; (b) determining that cells in thetest tissue sample express PD-L1 on the cell surface; (c) selecting ananti-PD-1 Ab as a treatment for the subject based on the recognitionthat an anti-PD-1 Ab is effective in patients whose test tissue samplecontains a proportion of cells above a predetermined threshold levelthat express PD-L1 on the cell surface; and (d) administering acomposition comprising a therapeutically effective amount of ananti-PD-1 Ab to the subject. In a further aspect, the disclosureprovides a method for treatment of a subject afflicted with cancer,which method comprises: (a) optionally providing a test tissue sampleobtained from a patient with cancer of the tissue, the test tissuesample comprising tumor cells and tumor-infiltrating inflammatory cells;(b) determining that cells in the test tissue sample do not expressPD-L1 on the cell surface; (c) selecting a standard-of-care therapeuticother than an anti-PD-1 Ab as a treatment for the subject based on therecognition that an anti-PD-1 Ab is ineffective in patients whose testtissue sample contains a proportion of cells that express PD-L1 on thecell surface is below a predetermined threshold level; and (d)administering the standard-of-care therapeutic to the subject.

This disclosure also provides a method of selecting an immunotherapy fora subject afflicted with cancer, which method comprises: (a) assayingcells of a test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells to assess the proportion of cellsin the test tissue sample that express PD-L1; and (b) based on anassessment that the proportion of cells that express membranous PD-L1 isabove a predetermined threshold level, selecting an immunotherapycomprising a therapeutically effective amount of an anti-PD-1 Ab for thesubject. The disclosure further provides a method of selecting atreatment for a subject afflicted with cancer, which method comprises:(a) assaying cells of a test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells to assess the proportion of cellsin the test tissue sample that express PD-L1; and (b) based on anassessment that the proportion of cells that express membranous PD-L1 isbelow a predetermined threshold level, selecting a standard-of-caretreatment other than an anti-PD-1 Ab for the subject.

In addition, the disclosure provides a method for treatment of a subjectafflicted with cancer, which method comprises administering to thesubject a composition comprising a therapeutically effective amount ofan anti-PD-1 Ab, the subject having been selected on the basis that theproportion of cells in a test tissue sample from the subject thatexpress PD-L1 is determined to exceed a predetermined threshold level,wherein the test tissue sample comprises tumor cells andtumor-infiltrating inflammatory cells. This disclosure also provides amethod for treatment of a subject afflicted with cancer, which methodcomprises administering to the subject a standard-of-care treatmentother than an anti-PD-1 Ab, the subject having been selected on thebasis that the proportion of cells in a test tissue sample from thesubject that express PD-L1 is determined to be below a predeterminedthreshold level, wherein the test tissue sample comprises tumor cellsand tumor-infiltrating inflammatory cells.

This disclosure further provides a method for selecting a cancer patientfor immunotherapy with an anti-PD-1 Ab, which method comprises: (a)optionally providing a test tissue sample obtained from a patient withcancer of the tissue, the test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells; (b) assaying the test tissuesample to determine the proportion of cells therein that express PD-L1on the cell surface; (c) comparing the proportion of cells that expressPD-L1 on the cell surface with a predetermined threshold proportion; and(d) selecting the patient for immunotherapy based on an assessment thatthe proportion of cells in the test tissue sample that express surfacePD-L1 is above the predetermined threshold level. In any of the methodsdescribed herein comprising a step for assessing PD-L1 expression, thetest tissue sample may be a FFPE tissue sample.

In addition, in any method where an immunotherapy is selected oradministered based on an assessment that the proportion of cells in atest tissue sample from the subject expresses PD-L1 at a level above apredetermined threshold level, it follows that a complementary method oftreatment may be performed wherein a standard-of-care treatment otherthan the immunotherapy is selected or administered based on anassessment that the proportion of cells in a test tissue sample from thesubject expresses PD-L1 at a level below the predetermined thresholdlevel.

This disclosure further provides a method for predicting the therapeuticeffectiveness of an anti-PD-1 Ab for treating a cancer patient, whichmethod comprises: (a) optionally providing a test tissue sample obtainedfrom a patient with cancer of the tissue, the test tissue samplecomprising tumor cells and tumor-infiltrating inflammatory cells; (b)assaying the test tissue sample to determine the proportion of cellstherein that express PD-L1 on the cell surface; (c) comparing theproportion of cells that express PD-L1 on the cell surface with apredetermined threshold value; and (d) predicting the therapeuticeffectiveness of the anti-PD-1 Ab, wherein if the proportion of cellsthat express PD-L1 on the cell surface exceeds the threshold proportionthe Ab is predicted to be effective in treating the patient, and whereinif the proportion of cells that express PD-L1 on the cell surface isbelow the threshold proportion the Ab is predicted to not be effectivein treating the patient.

This disclosure also provides a method for determining animmunotherapeutic regimen comprising an anti-PD-1 Ab for treating acancer patient, which method comprises: (a) optionally providing a testtissue sample obtained from a patient with cancer of the tissue, thetest tissue sample comprising tumor cells and tumor-infiltratinginflammatory cells; (b) assaying the test tissue sample to determine theproportion of cells therein that express PD-L1 on the cell surface; (c)comparing the proportion of cells that express PD-L1 on the cell surfacewith a predetermined threshold proportion; and (d) determining animmunotherapeutic regimen comprising an anti-PD-1 Ab based on thedetermination that the proportion of cells that express PD-L1 on thecell surface exceeds the predetermined threshold proportion.

Standard-of-Care Therapeutics

Several of the methods of treatment described herein comprise theadministration of a standard-of-care therapeutic to a patient. As usedherein, a “standard-of-care therapeutic” is a treatment process,including a drug or combination of drugs, radiation therapy, surgery orother medical intervention that is recognized by medical practitionersas appropriate, accepted, and/or widely used for a certain type ofpatient, disease or clinical circumstance. Standard-of-care therapeuticsfor treating different types of cancer are well known by persons ofskill in the art. For example, the National Comprehensive Cancer Network(NCCN), an alliance of 21 major cancer centers in the USA, publishes theNCCN Clinical Practice Guidelines in Oncology (NCCN GUIDELINES®) thatprovide detailed up-to-date information on the standard-of-caretreatments for a wide variety of cancers (see NCCN GUIDELINES®, 2013).By way of example, standard-of-care treatments for MEL, RCC and NSCLCare summarized below.

Melanoma

For in situ or early-stage MEL, surgical treatment is the primarytreatment. Where surgical excision is not feasible for in situ melanomadue to comorbidity or cosmetically sensitive tumor location, topicalimiquimod and radiotherapy are emerging as treatments, especially forlentigo maligna. Chemotherapeutic agents for treating MEL includedacarbazine, temozolomide, imatinib for melanoma with c-KIT mutation,high-dose interleukin-2, and paclitaxel with or without carboplatin.However, these treatments have modest success, with response rates below20% in first-line (1L) and second-line (2L) settings.

There is no consensus on the best treatments for metastatic melanoma,though a variety of treatments including excision to clear margins,intralesional injections, laser ablation, radiation and biochemotherapy(combination of chemotherapy and biological agents such asinterferon-alpha and IL-2) are being investigated. The therapeuticlandscape for metastatic melanoma has recently seen dramaticimprovements with the development of novel drugs such as vemurafenib andipilimumab. Vemurafenib specifically inhibits signaling by a mutatedintracellular kinase, BRAF, that is present in about 50% of patientswith metastatic melanoma (Flaherty et al., 2010). Ipilimumab is a HuMAbthat inhibits the immune checkpoint receptor, CTLA-4, and therebystimulates a T cell immune response (Hodi et al., 2010). Besides thesetwo agents, no other agent has demonstrated an OS benefit in a Phase 3randomized study. Dacarbazine is approved by the FDA and the EMA fortreatment of metastatic MEL with a reported objective response rate of5% to 20% and a median OS of 6.4 months, but these responses areshort-lived. Other drugs such as temozolomide and fotemustine have notresulted in significant improvement in survival when compared todacarbazine. IL-2 has also been approved by the FDA for the treatment ofmetastatic MEL as it is associated with a 15 to 20% response rateincluding 4-6% complete responses which can be durable, but it isassociated with significant toxicities including hypotension, cardiacarrhythmias, and pulmonary edema. Further details on standard-of-caretreatments for melanoma are provided by Garbe et al., 2012. Despite therecent approval of ipilimumab and vemurafenib for advanced MEL, there isstill a large unmet need for patients who have progressed on anti-CTLA-4therapy and a BRAF inhibitor (depending on BRAF status) or patients withpreviously untreated, unresectable or metastatic BRAF wild-type MEL. The5-year survival rate for late-stage MEL is currently only 15%.

Renal Cell Carcinoma

For clinically localized RCC (Stage IA and IB), surgical resection,including radical nephrectomy and nephron-sparing surgery, is aneffective therapy. Partial nephrectomy is generally not suitable forpatients with locally advanced tumors (Stage II and III), in which caseradical nephrectomy is preferred. Patients with Stage IV disease mayalso benefit from surgery, and cytoreductive nephrectomy before systemictherapy is recommended for patients with a potentially surgicallyresectable primary and multiple resectable metastases.

Until recently, the cytokines IL-2 and IFNα were the only activesystemic treatments for advanced or metastatic RCC. However, due to eachof these agent's limited clinical benefit and substantial toxicityprofile, newer targeted agents have largely replaced cytokines in thetreatment of advanced or metastatic renal cell carcinoma. Therecognition of the importance of hypoxia inducible factor alpha (HIFα)signaling in the pathogenesis of clear-cell RCC has led to widespreadstudy of two classes of targeted therapies, anti-angiogenic agents andmammalian target of rapamycin (mTOR) inhibitors (Mulders, 2009).Targeting of angiogenesis is rational because constitutive HIFαactivation leads to the upregulation or activation of several proteinsincluding vascular endothelial growth factor (VEGF), which cansubsequently lead to tumor proliferation and neovasculature formation.Targeting of the mTOR pathway is important because activation of theupstream PI3K/Akt/mTOR signaling pathway is one method by whichconstitutive HIFα activation or upregulation occurs (Mulders, 2009).Agents that target angiogenesis include VEGF-receptor (VEGFr) TKIs(e.g., sorafenib, sunitinib, pazopanib, axitinib, and tivozanib) andVEGF-binding mAbs (e.g., bevacizumab), while agents that target the mTORpathway include the mTOR inhibitors (e.g., everolimus and temsirolimus).However, most patients develop resistance, and overall survival (OS)improvement has only been shown in one Phase 3 trial in poor-riskpatients. Everolimus demonstrated a 3-month improvement in medianprogression-free survival (PFS) versus placebo, with no OS improvement(Motzer et al., 2008). Among the five approved anti-angiogenic agents(sorafenib, sunitinib, bevacizumab, pazopanib, and axitinib) and twoapproved mTOR inhibitors (temsirolimus, everolimus), only everolimus isapproved specifically for use after the failure of treatment withanti-angiogenic therapy. In the US, everolimus is indicated for thetreatment of advanced RCC after failure of treatment with sunitinib orsorafenib, whereas in the EU, everolimus is more broadly indicated forpatients with advanced RCC, whose disease has progressed on or aftertreatment with VEGF-targeted therapy.

Non-Small Cell Lung Cancer

NSCLC is the leading cause of cancer death worldwide, exceeding breast,colon and prostate cancer combined. The majority of subjects(approximately 78%) are diagnosed with advanced/recurrent or metastaticdisease. NSCLC therapies have incrementally improved OS, but benefit hasreached a plateau (median OS for late stage patients is just 1 year).Progression after 1 L therapy occurred in nearly all of these subjectsand the 5-year survival rate is only 3.6% in the refractory setting.

There is a particular unmet need among patients who have squamous cellNSCLC (representing up to 25% of all NSCLC) as there are few treatmentoptions after 1 L therapy. Surgery, radiation therapy (RT) andchemotherapy are the three modalities commonly used to treat NSCLCpatients. As a class, NSCLCs are relatively insensitive to chemotherapy,compared to small cell carcinoma. In general, for patients with Stage Ior II disease, surgery provides the best chance for cure, withchemotherapy increasingly being used both pre-operatively andpost-operatively. RT can also be used as adjuvant therapy for patientswith resectable NSCLC, the primary local treatment, or as palliativetherapy for patients with incurable NSCLC.

Patients with Stage IV disease who have a good performance status (PS)benefit from chemotherapy. Many drugs, including platinum agents (e.g.,cisplatin, carboplatin), taxanes agents (e.g., paclitaxel, albumin-boundpaclitaxel, docetaxel) vinorelbine, vinblastine, etoposide, pemetrexedand gemcitabine are useful for Stage IV NSCLC. Combinations using manyof these drugs produce 1-year survival rates of 30% to 40% and aresuperior to single agents. Specific targeted therapies have also beendeveloped for the treatment of advanced lung cancer. For example,bevacizumab (AVASTIN®) is a mAb that blocks vascular endothelial growthfactor A (VEGF-A). Erlotinib (TARCEVA®) is a small-molecule TKI ofepidermal growth factor receptor (EGFR). Crizotinib (XALKORI®) is asmall-molecule TKI that targets ALK and MET, and is used to treat NSCLCin patients carrying the mutated ALK fusion gene. Cetuximab (ERBITUX®)is a mAb that targets EGFR.

Squamous cell carcinoma (SCC) represents one quarter of NSCLC cases andhas limited treatment options. Currently, second-line treatment for SCCremains an area of unmet need. Single-agent chemotherapy is standard ofcare following progression with platinum-based doublet chemotherapy(Pt-doublet), resulting in median OS of approximately 7 months.Docetaxel remains the benchmark treatment in this line of therapyalthough erlotinib may also used with less frequency. Pemetrexed hasalso been shown to produce clinically equivalent efficacy outcomes butwith significantly fewer side effects compared with docetaxel in the 2Ltreatment of patients with advanced NSCLC (Hanna et al., 2004). Notherapy is currently approved for use in lung cancer beyond the 3Lsetting. Pemetrexed and bevacizumab are not approved in SCC, andmolecularly targeted therapies have limited application.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

Example 1 Cross-Competition Between Anti-PD-1 HuMAbs for Binding to CHOCells Expressing Human PD-1

Chinese Hamster Ovary (CHO) cells transfected to express human PD-1(CHO/PD-1 cells) were incubated with 10 μg/ml of Fab fragment of theanti-PD-1 HuMAb 5C4 or human IgG1 (hIgG1) isotype control Ab for 30minutes at 4° C. before addition of anti-PD-1 HuMAbs 2D3, 7D3 or 4H1 ata concentration of 0.2 μg/ml. Binding of 4H1, 2D3 or 7D3 to CHO/PD-1cells were detected by fluorescein isothiocyanate (FITC)-conjugated goatanti-hIgG, Fc-gamma specific Ab. In the case of cross-competition assaywith 5C4 and 17D8, CHO/PD-1 cells were incubated with the whole moleculeof 5C4 before addition of FITC-labeled 17D8. Binding of 2D3, 7D3, 4H1 or17D8 to the CHO/PD-1 cells was measured by flow cytometric analysisusing a FACS® calibur flow cytometer (Becton Dickinson, San Jose,Calif.).

The results are depicted in FIG. 1. The data show that the 5C4 Fabfragment substantially blocked the binding of mAbs 5C4 itself, as wellas the binding of 2D3, 7D3 (FIG. 1A) and 4H1 (FIG. 1B), while the 5C4whole mAb substantially blocked the binding of 17D8 (FIG. 1C) toCHO/PD-1 cells as measured by mean fluorescent intensity (MFI) ofstaining.

Example 2

Cross-Competition Between Anti-PD-L1 HuMAbs for Binding to CHO CellsExpressing Human PD-L1

CHO cells transfected to express hPD-L1 (CHO/PD-L1 cells) were incubatedwith 10 μg/ml of each of ten unconjugated human anti-PD-L1 mAbs (5F8,7H1, 10H10, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7, and 13G4) or human IgG1(hIgG1) isotype control Ab for 20 min at 4° C. FITC-conjugated 10H10(A), 3G10 (B), 10A5 (C), 11E6 (D), 12A4 (E) or 13G4 (F) was added to thecells to a final concentration of 0.09 μg/ml (B, D), 0.27 μg/ml (A, C),0.91 μg/ml (F), or 2.73 μg/ml (E) for an additional 20 min at 4° C.without prior washout of unbound, unconjugated Ab. Different quantitiesof the various FITC-conjugated HuMAbs were used due to differences inbinding efficiency following labeling, and the optimal amounts of theseFITC-conjugated HuMAbs were previously determined by dose-titrationanalysis of binding to CHO/PD-L1 cells. Binding of FITC-conjugated10H10, 3G10, 10A5, 11E6, 12A4 or 13G4 to the CHO/PD-L1 cells wasmeasured by flow cytometry.

The results are depicted in FIG. 2. Binding of labeled 10H10 waspartially blocked by 10A5, 11E6 and 13G4, but was substantially blockedonly by itself (FIG. 2A). Conversely, 10H10 substantially blocked thebinding only of itself to CHO/PD-L1 cells. Each of anti-PD-L1 HuMAbs5F8, 7H1, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7 and 13G4 substantiallyblocked binding of labeled mAbs 3G10 (FIG. 2B), 10A5 (FIG. 2C), 11E6(FIG. 2D), 12A4 (FIG. 2E) and 13G4 (FIG. 2F) to CHO/PD-L1 cells asmeasured by MFI, though mAbs 5F8 and 13G4 generally blocked binding ofthe labeled mAbs to a slightly lesser extent.

Example 3

Cross-Competition Between Anti-PD-L1 mAbs for Binding to OvarianCarcinoma Cells Expressing Human PD-L1

Anti-PD-L1 HuMAbs 5F8, 12B7, 3G10, 1B12, 13G4, 10H10, 10A5 and 12A4, anda human IgG1 (huIgG1) isotype control Ab were serially diluted from 10μg/ml and incubated with hPD-L1-expressing ES-2 ovarian carcinoma cellsfor 20 minutes at 4° C. Without washing, biotinylated-12A4 Ab was addedto a final concentration of 0.4 μg/ml for an additional 20 minutes at 4°C. After washing, bound biotin-12A4 was detected using fluorescentstreptavidin-PE secondary reagent and measured by flow cytometry. FIG. 3shows the fluorescence of bound biotin-12A4 plotted against theconcentration of unlabeled hPD-L1 HuMAbs. Binding of biotin-12A4 to ES-2cells was substantially blocked by 12A4 itself and by 1B12 and 12B7, andwas moderately to significantly blocked by mAbs 5F8, 10A5, 13G4 and3G10, but was not blocked by mAb 10H10.

Example 4 Design of Phase I Clinical Study of Anti-PD-1 Antibody

A Phase I study was conducted to assess the safety, antitumor activity,and pharmacokinetics of an anti-PD-1 in patients with selected advancedsolid tumors. The human anti-PD-1 mAb, BMS-936558 (also referred toherein as nivolumab, and in U.S. Pat. No. 8,008,449 as 5C4), wasadministered as an intravenous infusion every 2 weeks of each 8-weektreatment cycle. Patients continued treatment for up to 2 years (12cycles), unless they experienced complete remission, unacceptabletoxicity, disease progression, or withdrew consent. In patients who wereotherwise clinically stable, study treatment was continued beyondapparent initial disease progression until further progression was notedas recommended by proposed immune response criteria (Wolchok et al.,2009). Patients with stable disease (SD) or an ongoing objectiveresponse (OR: complete [CR] or partial response [PR]) at the end oftreatment were followed for 1 year and were offered retreatment for 1additional year in the event of progression.

Dose Escalation

Patients with advanced melanoma (MEL), non-small cell lung (NSCLC),renal cell carcinoma (RCC), castration-resistant prostate (CRPC) andcolorectal cancer (CRC) were eligible to enroll. Cohorts of 3-6 patientsper dose level were enrolled sequentially at 1.0, 3.0, and 10.0 mg/kg.Dose escalation proceeded when a minimum of 3 patients had completed thesafety evaluation period (56 days) at the given dose level, withdose-limiting toxicity in less than one-third of patients. Intra-patientdose escalation was not permitted.

Cohort Expansion

A maximum tolerated dose (MTD) was not reached. Initially, 5 expansioncohorts of approximately 16 patients each were enrolled at 10 mg/kg forMEL, NSCLC, RCC, CRPC and CRC. Based on initial signals of activity,additional expansion cohorts of approximately 16 patients each wereenrolled for MEL (at 1.0 and 3.0 mg/kg, followed by cohorts randomizedto 0.1, 0.3, or 1.0 mg/kg), NSCLC (squamous or nonsquamous histologycohorts randomized to 1, 3, or 10 mg/kg), and RCC (at 1.0 mg/kg).

Patients

Eligible patients had documented advanced solid tumors; age >18 years;life expectancy >12 weeks; Eastern Cooperative Oncology Groupperformance status of ≤2; measurable disease by Response EvaluationCriteria in Solid Tumors (RECIST), v1.0 with modification (see Topalianet al., 2012b); adequate hematologic, hepatic, and renal function; andreceived 1-5 prior systemic treatment regimens. Patients with stabletreated brain metastases were enrolled. Exclusion criteria included ahistory of chronic autoimmune disease, prior therapy with T-cellmodulating Abs (e.g., anti-CTLA-4), conditions requiringimmunosuppressive medications, and chronic infections (HIV, hepatitis Bor C).

A total of 296 patients with advanced solid tumors including MEL(n=104), NSCLC (n=122), RCC (n=34), CRPC (n=17), and CRC (n=19) weretreated with BMS-936558 for 40 months up to February 2012. By March2013, 304 patients including patients with non-small cell lung cancer(n=127), melanoma (n=107), RCC (n=34), CRPC (n=17), and CRC (n=19) hadbeen treated with BMS-936558 from October 2008 through March 2012, allwith a minimum of one year and up to about 4.4 years of observation. Twopatients did not receive a full cycle of treatment and were notconsidered response-evaluable. The majority of patients were heavilypretreated, with 47% having received at least 3 prior regimens. Notableprior therapies included immunotherapy (64%) and B-RAF inhibitor (8%) inMEL patients; platinum-based chemotherapy (94%) and tyrosine kinaseinhibitors (TKIs, 34%) in NSCLC patients; and nephrectomy (94%),immunotherapy (59%), and anti-angiogenic therapy (74%) in RCC patients.Baseline characteristics of the total treated population (N=296) weresimilar to those of the efficacy population (response evaluablepatients, N=236). Details on the patient pre-treatments are provided inTopalian et al., 2012b.

Statistical Analysis

All patients (N=304) treated as of the date of analysis (March 2013)were used for summaries of baseline characteristics and AEs.Pharmacokinetic and molecular-marker populations consisted of treatedpatients with available data as of the date of analysis. The efficacypopulation consisted of response-evaluable patients commencing treatmentat least 8 months before the date of analysis. Tumor measurements werecollected after each treatment cycle (4 doses) by investigators.Individual best objective responses based on the tumor measurements wereassessed by the sponsor per modified RECIST v1.0.

Objective response was confirmed by at least one sequential tumorassessment. Objective response and stable disease rates were estimatedwith confidence intervals using the Clopper-Pearson method.Time-to-event endpoints including progression-free survival, overallsurvival, survival rates, and duration of response, were estimated usingthe Kaplan-Meier method. AEs were coded using Medical Dictionary forRegulatory Activities (MedDRA), version 14.1. AEs of special interest(AEOSIs), with potential immune-related etiologies, defined as adverseevents that require more frequent monitoring and/or unique intervention,were identified using a pre-defined list of MeDRA terms. Individual bestORs were derived from investigator-reported data per modified RECISTv1.0. OR was confirmed by at least one sequential tumor assessment andOR rate (ORR={[CR+PR]÷n}×100) was calculated.

Example 5

Safety Evaluations on Patients Treated with Anti-PD-1 Antibody

Safety evaluations, including clinical examination and laboratoryassessments, were conducted in all treated patients at baseline andregular intervals up to 100 days following last administration of drug.The severity of AEs was graded based on the NCI Common TerminologyCriteria for Adverse Events (NCI CTCAE), v3.0. Computed tomography (CT)or magnetic resonance imaging was performed for tumor assessment atbaseline and following each treatment cycle.

A MTD was not defined across the doses of BMS-936558 tested on thisstudy. A relative BMS-936558 dose intensity of 90% or higher wasachieved in 87% of patients (see Topalian et al., 2012b, for details).AEs were coded using Medical Dictionary for Regulatory Activities(MedDRA), version 14.1. AEOSIs were identified using a pre-defined listof MeDRA terms. Fifteen of 296 (5%) patients discontinued treatment dueto BMS-936558-related AEs. As of the date of analysis, 62 (21%) patientshad died, with disease progression being the most common cause of death(Topalian et al., 2012b).

The most common adverse events, regardless of causality includedfatigue, decreased appetite, diarrhea, nausea, cough, dyspnea,constipation, vomiting, rash, pyrexia and pruritus (Topalian et al.,2012b). Common BMS-936558-related AEs included fatigue, rash, diarrhea,decreased appetite, and nausea. The majority of the events were lowgrade, with grade 3-4 drug-related AEs observed in 41 of 296 (14%)patients. Drug-related serious AEs occurred in 32 of 296 (11%) patients(Topalian et al., 2012b). The spectrum, frequency, and severity ofBMS-936558-related AEs were generally similar across the dose levelstested. Drug-related AEOSIs, with potential immune-related etiologies,included pneumonitis, vitiligo, colitis, hepatitis, hypophysitis, andthyroiditis among others. Hepatic or gastrointestinal AEOSIs weremanaged with treatment interruption and, as necessary, withadministration of corticosteroids. Among patients treated to-date, theseAEs (in 13 patients with diarrhea; 11 patients with hepatic AEs) werereversible in all cases. Endocrine AEOSIs were managed with replacementtherapy. At the discretion of the treating physician, patientssuccessfully reinitiated treatment with BMS-936558. Drug-relatedpneumonitis occurred in 9 of 296 (3%) patients. Grade 3-4 pneumonitisdeveloped in 3 patients (1%). No clear relationship between theoccurrence of pneumonitis and tumor type, dose level, or the number ofdoses received was noted. Early grade pneumonitis was generallyreversible with treatment discontinuation and corticosteroidadministration. In 3 patients with pneumonitis, infliximab and/ormycophenolate were utilized for additional immunosuppression; however,given the small number of patients and variable outcomes, theeffectiveness of this treatment is unclear. There were 3 (1%)drug-related deaths due to pneumonitis (2 NSCLC patients, 1 CRC).

Example 6 Pharmacokinetics/Pharmacodynamics Analyses on Anti-PD-1Antibody

For pharmacokinetic (PK) analyses, serial blood samples were collectedand serum concentrations of BMS-936558 were quantified using by ELISA.For pharmacodynamic (PD) analysis, peripheral blood mononuclear cellswere isolated from patients at baseline and following cycle 1 toestimate PD-1 receptor occupancy (RO) by BMS-936558 on circulating CD3+T-cells via flow cytometry (Brahmer et al., 2010).

The maximum concentration of BMS-936558 was observed at a median T. of1-4 hours after the start of infusion. The PK of BMS-936558 was linearwith a dose proportional increase in C_(max) and AUC_((0-14 d)) in thedose range of 0.1-10 mg/kg (n=35). BMS-936558 PD was assessed by PD-1 ROon circulating T-cells. PBMCs from 65 MEL patients treated with onecycle of BMS-936558 at 0.1-10 mg/kg biweekly demonstrated medianoccupancy of PD-1 molecules on circulating CD3⁺ T-cells by BMS-936558ranging from 64%-70% (see Topalian et al., 2012b, for details).

Example 7 Antitumor Efficacy Exhibited by Anti-PD-1 Antibody

Analysis of data obtained since February 2012 is ongoing; thus, unlessotherwise noted, the data presented below were obtained as of February2012. Clinical antitumor activity was observed at all BMS-936558 dosestested. ORs (confirmed CR or PR) were observed in a substantial portionof patients with NSCLC, MEL, and RCC (Tables 1-3; FIG. 4), and invarious sites of metastatic disease including liver, lung, lymph nodes,and bone (FIGS. 5-7 and not shown). Tumor regressions followedconventional as well as “immune-related” patterns of response, such asprolonged reduction in tumor burden in the presence of new lesions.Individual best overall responses were derived frominvestigator-reported data according to modified RECIST v1.0. OR wasconfirmed by at least one sequential tumor assessment. At the time ofdata analysis, 2 patients with NSCLC who were treated with 10 mg/kg hadunconfirmed responses, and 8 additional patients (with MEL, NSCLC, orRCC) had a persistent reduction in baseline target lesions in thepresence of new lesions, (i.e., an “immune-related” response pattern).None of these patients was categorized as a responder for the purpose ofcalculating OR rates. Antitumor responses and/or prolonged diseasestabilization were observed in patients irrespective of prior therapiesreceived (see summary of progression free interval for patients with ORand SD in Supplementary Appendix 4 of Topalian et al., 2012b).

In NSCLC patients, 14 ORs were observed at BMS-936558 doses of 1, 3, or10 mg/kg with response rates of 6%, 32%, and 18%, respectively. ORs wereobserved across NSCLC histologies: 6 responders of 18 squamous (33%), 7responders of 56 nonsquamous (13%), and 1 of 2 unknown. All 14 patientswith ORs started treatment ≥24 weeks before data analysis, and of these,8 had response duration ≥24 weeks (Table 1). Stable disease (SD) lasting≥24 weeks was observed in 5 (7%) NSCLC patients, all with nonsquamoushistology. Among MEL patients, 26 ORs were observed at doses rangingfrom 0.1-10 mg/kg, with response rates ranging from 19%-41% per doselevel. At the 3 mg/kg dose level, ORs were noted in 7 of 17 (41%)patients. Of 26 MEL patients who achieved an OR, 17 started treatment ≥1year before data analysis, and of these, 13 patients had an OR duration≥1 yr. The remaining 8 patients with OR were on study <1 year and 6 hadresponses ranging from 1.9-5.6 months. SD lasting ≥24 weeks was observedin 6 (6%) patients. In RCC patients, ORs occurred in 4 of 17 (24%)patients treated with a BMS-936558 dose of 1 mg/kg and 5 of 16 (31%)patients treated with 10 mg/kg. Among 8 RCC patients with OR who startedtreatment ≥1 year prior to data analysis, 5 (63%) had OR duration ≥1 yr.SD lasting ≥24 weeks was observed in an additional 9 (27%) patients.

TABLE 1 Clinical Activity of BMS-936558 in the Efficacy Population* (N =236)^(†) SD ≥24 wk Dose ORR^(‡) No. PFSR^(§) at (mg/ No. Patients (%)Patients (%) 24 wk (%) Tumor Type kg) n [95% CI]^(†) [95% CI] [95% CI]MEL 0.1 14  4 (29) [8-58] 1 (7) [0.2-34] 40 [13-66] 1.0 27  8 (30)[14-50] 3 (11) [2-29] 45 [26-65] 3.0 17  7 (41) [18-67] 1 (6) [0.1-29]55 [30-80] 10.0 20  4 (20) [6-44] 0 30 [9-51]  ALL MEL 94 26 (28)[19-38] 6 (6) [2-13] 41 [30-51] NSCLC** All 1 18  1 (6) [0.1-27] 1 (6)[0.1-27] 16 [0-34]  Squamous 1 5 0 0 0 Non- 1 12 0 1 (8) [0.2-39] 14[0-37]  squamous Unknown 1 1  1(100) [3-100] 0 1 All 3 19  6 (32)[13-57] 2 (11) [1-33] 41 [18-64] Squamous 3 6  3 (50) [12-88] 0 50[10-90] Non- 3 13  3 (23) [5-54] 2 (15) [2-45] 37 [10-64] squamous All10 39  7 (18) [8-34] 2 (5) [0.6-17] 24 [11-38] Squamous 10 7  3 (43)[10-82] 0 43 [6-80]  Non- 10 31  4 (13) [4-30] 2 (7) [0.8-21] 21 [6-36] squamous Unknown 10 1 0 0 0 ALL 76 14 (18) [11-29] 5 (7) [2-15] 26[16-36] NSCLC All 18  6 (33) [13-59] 0 33 [12-55] Squamous All 56  7(13) [5-24] 5 (9) [3-20] 22 [11-34] Non- squamous RCC 1 17  4 (24)[7-50] 4 (24) [7-50] 47 [23-71] 10 16  5 (31) [11-59]^(‡) 5 (31)[11-59]^(¶) 67 [43-91] ALL RCC 33  9 (27) [13-46] 9 (27) [13-46] 56[39-73  *The efficacy population consists of response-evaluable patientswhose treatment was initiated at least 8 months before data analysis inFebruary 2012, and had measurable disease at baseline and one of thefollowing: at least 1 on-treatment scan or clinical evidence of diseaseprogression or death. ^(†)CR denotes complete response, MEL melanoma,NSCLC non-small cell lung cancer, ORR objective response rate, PFSRprogression-free survival rate, PR partial response, RCC renal cellcancer, SD stable disease, n number of patients. ^(‡)Objective responserates ({[CR + PR] ÷ n} × 100) have been calculated based on confirmedresponses with confidence intervals calculated using the Clopper-Pearsonmethod. Individual patient responses were adjudicated per RECIST v1.0with modification (see Topalian et al., 2012b). ^(§)Progression-freesurvival rate was the proportion of patients who did not progress andwere alive at 24 weeks calculated by the Kaplan-Meier methodology withconfidence intervals using the Greenwood method. ^(¶)One CR. **One NSCLCpatient who was treated at the 3 mg/kg dose level had an initialevaluation of progressive disease, subsequently had a PR, and wasclassified as a responder.

TABLE 2 Clinical Activity of BMS-936558 in the Efficacy Population*ORR^(a) SD ≥24 wk No. of No. of patients/total patients/total Median no.of no. of Overall Overall Survival Rate Tumor patients (%) patients (%)Survival (95% CI); patients at risk, n Type [95% CI] [95% CI] (95% CI) 1y 2 y 3 y NSCLC 20/122 (16.4)  11/122 (9.0)   NA 43 32 24 [10.3, 24.2] [4.6, 15.6] (33-53); (18-47); 4 (7-41); 1 24 Squamous  9/48 (18.8) NANA NA NA NA  [8.9, 32.6] Non-sq. 11/73 (15.1) NA NA NA NA NA  [7.8,25.4] MEL 33/106 (31.1)  6/106 (5.7)  16.8 61 44 40 [22.5, 40.9]  [2.1,11.9] (12.5, (52-71); (33-56); (29-52); 1 NR) 50 24 RCC 10/34 (29.4)9/34 (26.5) NR 70 52 52 [15.1, 47.5] [12.9, 44.4] (13.6, (55-86);(32-72); 7 (32-72); 1 NR) 22 *The efficacy population consists ofresponse-evaluable patients up to March 2013 ^(a)Objective response ratewas calculated as {[CR + PR] ÷ n} × 100. NR—not reached NA—not availablefrom Jul. 3, 2012 data cut. The percentage of patients with stabledisease for ≥36 weeks or ≥48 weeks [95% CI], median duration of response(months) and median progression-free survival (95% CI) were N/A.

The following results are drawn from preliminary analyses of BMS-936558clinical efficacy data as of March 2013. Sustained survival, asreflected by median overall survival and 1- and 2-year landmark overallsurvival rates, was noted in each of the responding patient populations(Table 2). Median overall survivals of 9.6 months for lung cancer, 16.8months for melanoma, and greater than 22 months for kidney cancer, wereobserved. Thirteen patients with melanoma, lung or kidney cancer werecharacterized as having unconventional response patterns that did notmeet RECIST criteria (e.g., persistent reduction in target lesions inthe presence of new lesions or following initial progression) (Wolchoket al, 2009).

TABLE 3 Duration of Objective Responses to BMS-936558* Tumor Dose No. ofPatients Type (mg/kg) with OR Duration of Response (months)^(†) MEL 0.14 7.5+, 5.6+, 5.6, 5.6 0.3 3 3.8+, 2.1+, 1.9+ 1 8 24.9+, 22.9, 20.3+,19.3+, 18.4+, 7.6+, 5.6+, 5.3+ 3 7 22.4+, 18.3+, 15.2+, 12.9, 11.1, 9.3,9.2+ 10 4 24.6+, 23.9+, 18.0+, 17.0 NSCLC^(§) 1 1 9.2+ 3 6 30.8+, 7.6+,5.5+, 3.7+, 1.9+, NA^(‡) 10 7 14.8+, 7.6+, 7.3+, 6.7, 4.2, 3.7+, 3.7 RCC1 4 17.5+, 9.2+, 9.2, 5.6+ 10 5 22.3+. 21.7+, 12.9, 12.0, 8.4 *MELdenotes melanoma, NA not applicable, NSCLC non-small cell lung cancer,RCC renal cell cancer. ^(†)Time from first response to time ofdocumented progression, death, or for censored data, time to last tumorassessment. ^(‡)One patient was treated beyond an initial evaluation ofprogressive disease and subsequently had a PR; this patient wasclassified as a responder for the purposes of calculating response ratesby RECIST v1.0 but was not eligible for calculation of duration ofresponse.

Example 8 Measurement of Membranous PD-L1 Expression by Standard IHCAssay

IHC staining of PD-L1 was performed on pretreatment formalin-fixedparaffin-embedded (FFPE) tumor specimens using the murine anti-humanPD-L1 mAb 5H1 (Dong et al., 2002) in a standard IHC protocol (Taube etal., 2012; Supp. Materials). Briefly, 5 μm-FFPE sections mounted onglass slides were deparaffinized in xylene and antigen retrieval wasperformed using a Tris-EDTA buffer, pH 9.0 at 120° C. for 10 min in aDecloaking Chamber (Biocare Medical). Endogenous peroxidase, biotin andproteins were blocked (CAS system K1500, Dako; Avidin/biotin BlockingKit, SP-2001, Vector Laboratories; Serotec Block ACE). The primary 5H1Ab was applied at a concentration of 2 μg/ml and allowed to incubate at4° C. for 20 h. Secondary Ab (biotinylated anti-mouse IgG1, 553441 BD)was applied at a concentration of 1 μg/ml for 30 min at room temperature(RT). The signal was then developed with amplification according to themanufacturer's protocol (CAS system K1500, Dako). Sections werecounterstained with hematoxylin, dehydrated in ethanol and cleared inxylene, and a coverslip was applied.

The percentage of tumor cells exhibiting cell surface staining for PD-L1was scored by two independent pathologists who were blinded to treatmentoutcomes. PD-L1 positivity was defined per specimen by a 5% expressionthreshold (Taube et al., 2012; Thompson et al., 2006), and in cases withmultiple specimens, if any specimen met this criterion. A Fisher's exacttest was applied to assess the association between PD-L1 expression andOR, noting, however, that this analysis was based in part on optionalbiopsies from a non-random subset of the population and testing of astatistical hypothesis was not pre-specified.

Sixty-one pretreatment tumor specimens from 42 patients (18 MEL, 10NSCLC, 7 CRC, 5 RCC, and 2 CRPC) were analyzed for tumor cell surfacePD-L1 expression (FIG. 8). Biopsy specimens from 25 of 42 patients werepositive for PD-L1 expression by IHC. A Fisher's exact test was appliedto assess the association between PD-L1 expression and OR in a post-hocanalysis. Among the 42 surface-PD-L1⁺ patients, 9 (36%) achieved an OR.Importantly, among 17 patients with PD-L1⁻ tumors, none achieved an OR.Thus, in a subset of patients cell surface expression of PD-L1 on tumorcells in pretreatment biopsies is associated with an increased rate ofOR among patients treated with BMS-936558, while no patients withdocumented PD-L1-negative tumors experienced an OR. These data indicatethat tumor PD-L1 expression is a molecular marker that can enablepatient selection for anti-PD-1 immunotherapy.

Example 9

Isolation of Rabbit mAbs that Detect Membranous hPD-L1 Antigen in FFPETissues

Rabbit Abs against human PD-L1 polypeptide were prepared by Epitomics,Inc. (Burlingame, Calif.) by immunization of rabbits using a recombinanthuman PD-L1 fusion protein. Antiserum titers were evaluated usingstandard direct ELISA with the hPD-L1 antigen and cell ELISA usingtransfected cells overexpressing hPD-L1. These Abs were also screenedfor their ability to bind to PD-L1 by IHC assay of FFPE tissue sections.The rabbit with the highest Ab titer was selected for splenectomy.Lymphocytes isolated from the spleen were fused to myeloma cells in40×96-well plates, and screened by ELISA against the immunizing PD-L1antigen and by cell ELISA against cells overexpressing hPD-L1. Positiveclones were expanded into 24-well plates, and confirmatory screens wereconducted by direct ELISA and cell ELISA. The supernatants (sups) ofclones that were specific to the screening antigen were re-screened byIHC.

A set of mouse anti-hPD-L1 mAbs were also produced by immunization ofmice using a protocol similar to that described above for the rabbitmAbs.

Out of a total of 185 multiclones from both rabbit and mouseimmunizations screened, only ten rabbit multiclone Abs specificallydetected the membranous form of hPD-L1. None of the purified mousesubclones were found to specifically detect cell surface hPD-L1. Sixtysubclones from the top five rabbit multiclones (designated Nos. 13, 20,28, 29 and 49, each comprising 12 subclones) were screened initially byIHC on FFPE low density tissue microarrays (TMAs), followed byconfirmation and specificity verification in narrowed 25 subclones.Rabbit IgG was used as a negative isotype control, and mAb 5H1 (Dong etal., 2002) was used as the positive control. Specificity was furtherverified by antigen preabsorption assay. Through two rounds of IHC, thefollowing 15 purified subclones were selected as the most promising Absin terms of specificity and intensity of staining: 13-1, 13-3, 13-7,13-8; 20-5, 20-7, 20-12, 20-6; 28-1, 28-8, 28-12; 49-5, 49-7, 49-9; and29-8 Immunoreactivity data on these selected Abs are summarized in Table4.

Additional assays were performed to further characterize the purified Abclones, including determining binding affinity and cross-competitionamong the Abs (to identify overlapping versus different epitope regions)by surface plasmon resonance. All the Abs exhibited high bindingaffinity (K_(D)<10⁻⁹ M). These 15 purified clones were also re-screenedby IHC on FFPE low density TMA or regular sections against various celland tissue types known to be positive or negative for cell surfaceexpression of PD-L1. Rabbit IgG was used as the isotype control, and mAb5H1 was used as the positive control. At high concentration (10 μg/ml),clones 28-x and 49-x displayed low to moderate levels of backgroundstaining in tissues, while clones 13-x exhibited no background staining,which suggests that the 13-x clones have a wider dynamic range. The 20-xclones displayed various degree of background staining which wasprimarily cytoplasmic and diffuse. The clone with most robust detectionspecifically of membranous PD-L1, rabbit clone 28-8 (K_(D)=100 p M, asdetermined by SPR), was selected as the lead Ab for subsequent IHCassays. MAbs 28-1, 28-12, 20-12 and 29-8 had K_(D) values of 130 pM, 94pM, 160 pM and 1200 pM, respectively. The sequences of the V_(H) andV_(κ) regions of mAb 28-8 are set forth in SEQ ID NOs. 35 and 36,respectively. The 28-8 Ab was shown to recognize a different epitopefrom mouse mAb 5H1, based on SPR analysis. Clones 28-1, 28-12, 29-8 and20-12 were the next best Abs in terms of robust detection of membranousPD-L1 in FFPE tissues. Although mAb 13-1 had the best specificity interms of detection of membranous PD-L1, the maximal detection level waslower than that of the other lead Abs. Western blotting was alsoperformed with plus/minus antigen competition to verify the specificityof the top selected Abs for PD-L1.

TABLE 4 Immunoreactivity of Rabbit Anti-hPD-L1 mAbs Non-Specificstaining Specific Staining Background Pos. vs Neg. Intensity Range onStaining on Antibody Staining on PD- Tissues^(†) (Very High, Tissues(High, Name L1 Cells* High, Medium, Low) Medium, Low) 13-1 Pos Low toVery High None 13-3 Pos Low to High None 13-7 Pos Low to High None 13-8Pos Low to High None 20-5 Pos Low to Very High High 20-6 Pos Low to HighMedium 20-7 Pos Low to High Medium 20-12 Pos Low to High Medium 28-1 PosLow to Very High None 28-8 Pos Medium to Very High None 28-10 Pos Mediumto Very High Low 28-12 Pos Low to Very High None 49-5 Pos Low to HighNone 49-7 Pos Low to High Low 49-9 Pos Low to High None 5H1 Pos High toVery High None Rb IgG Neg Neg None *PD-L1 stably transfected CHO cellsvs. CHO—S control; ^(†)PD-L1 positive tissues included placenta and onenon-small cell lung cancer; Detection up to “very high” expressionsuggests better sensitivity at detecting membranous PD-L1.

The binding of mAbs 5H1 and 28-8 to membranous PD-L1 in FFPE test tissuesamples comprising tumor cells and tumor-infiltrating inflammatory cellsfrom different types of tumors was compared. Membranous PD-L1 expressionwas evaluated using the histoscore method performed by 2 independentpathologists. Four NSCLC, 2 MEL, and 2 RCC tumors were stained with 28-8at 2 mg/ml and 5H1 at 5 mg/ml. The data are tabulated in Table 5, andshown graphically in FIG. 9. The rabbit mAb 28-8 showed better detection(higher histoscores) for 7 out of 10 samples using 2.5-fold less Ab, andin only one sample was the histoscore for 5H1 slightly higher than formAb 28-8.

TABLE 5 Comparison of mAbs 28-8 and 5H1 by histoscore analysisHistoscore Histoscore Tissue Sample I.D. Average (5H1) Average (28-8)NSCLC 1080754B 245 261 NSCLC 1080766B 103 130 NSCLC 1080790 40 37 NSCLC1080993B 12 16 Mel T030668 98 113 Mel T980744 98 123 Mel 1168657B 0 0Mel T980747 1 9 RCC 1164619B 4 4 RCC 1167809B 108 125

Taube et al., 2012 demonstrated by flow cytometry on cultured cells thatmAb 5H1 bound to the cell surface, and the specificity of binding toPD-L1 was confirmed using a PD-L1 fusion protein to competitively blockbinding of the 5H1 mAb to tissue sections. These authors also compared5H1 with a rabbit polyclonal anti-hPD-L1 Ab, 4059, previously describedby Gadiot et al., 2011, and found that whereas 5H1 showed a cell surfacestaining pattern on FFPE samples, Ab 4059 demonstrated diffusecytoplasmic staining. Further, when 5H1 was compared to Ab 4059 bywestern blot analysis, Ab 4059 bound to multiple proteins in lysates ofmelanoma cells in addition to a 50 kDa protein corresponding to theexpected mass of glycosylated PD-L1, in contrast to 5H1 whichspecifically detected the 50 kDa band of glycosylated PD-L1 (Taube etal., 2012).

In the present study, an automated IHC assay (see Example 10) was usedto evaluate the binding of several commercially available anti-PD-L1 Absand 5H1 (Dong et al., 2002) to FFPE tissue samples containing variouscells expressing PD-L1. The results, summarized in Table 6, show thatnone of the commercially available Abs tested specifically recognizedmembranous PD-L1 expression in human tissues known to express PD-L1, orto clearly distinguish CHO cells expressing PD-L1 versus theuntransfected parent CHO cells that did not express PD-L1. The inabilityof the polyclonal Ab (pAb) 4059 to bind specifically recognizedmembranous PD-L1 is consistent with the findings of Taube et al., 2012.The binding of 28-8 was similar to that of 5H1 in this assay, thoughhistoscore analysis suggests that 28-8 performs better than 5H1.

TABLE 6 Binding of mAbs to FFPE Samples Containing PD-L1-expressingcells Clone No. (mAb)/ Pos. vs. Neg. Human Antibody Catalog No. Stainingon Positive Source Types (pAb) PD-L1 Cells* Tissues^(†) MBL mAb 27A2Failed Failed BioLegend mAb 29E.2A3 Failed Failed eBiosciences mAb M1H1No Staining No staining Collaborator mAb 5H1 Passed Passed ProSci pAb4059 Failed Failed LifeSpan pAb LS-B480/0604 Failed Failed BioSciences*PD-L1 stably transfected CHO cells vs. parent CHO—S negative control;^(†)PD-L1 positive tissues included tonsil and/or thymus; mAb, mousemAb; pAb, rabbit polyclonal Ab.

Example 10 Development of Automated IHC Protocol for Assessing PD-L1Expression

An automated IHC protocol was developed to assay PD-L1 expression inFFPE specimens. Tissue sections (4 μm) were mounted on slides,deparaffinized in an autostainer (Leica) by soaking twice for 5 min inxylene, and re-hydrated through soaking twice for 2 min each time in100% EtOH, twice in 95% (v/v) EtOH, once in 70% (v/v) EtOH, and once inde-ionized water (dH₂O). Antigen retrieval was performed using adecloaking chamber (Biocare Medical Decloaking Chamber Plus) and Dako pH6 buffer, heated to 110° C. (P1) for 10 min, then moved to the next step(P2 FAN ON at 98° C.; FAN OFF at 90° C.). The slides were cooled at roomtemperature (RT) for 15 min and rinsed with water for about 1 min.

Reagents were set up on an autostainer (BIOGENEX® i6000), using a pappen to define the tissue area. The IHC assay, run using the autostainerin research mode, comprised the following steps: neutralizing endogenousperoxidase using the Peroxidase Block (Leica) for 10 min, followed byrinsing 3 times with IHC wash buffer (Dako); applying Protein Block(Leica) to the slides, and incubating for 10 min at RT, followed bywashing 3 times with wash buffer; applying the primary Ab to the slides(2 μg/ml) and incubating for 1 h at RT, followed by washing 6 times withwash buffer; adding Post Primary Block (NovoLink Kit) to the slides andincubating for 30 min, followed by washing 6 times with wash buffer;adding NovoLink Polymer (NovoLink Kit) to the slides and incubating for30 min, followed by washing 6 times with wash buffer; adding the DABchromogen substrates (NovoLink Kit) and developing for 3 min, followedby rinsing 5 times with dH₂O at RT; counterstaining with hematoxylin(NovoLink Kit) for 1 min at RT, followed by washing 3 times with dH₂Ofor 5 times at RT. The primary Ab was selected from the rabbitanti-PD-L1 Abs shown in Table 4; mAb 28-8 was the preferred Ab. As anegative control, rabbit IgG (Dako) was used. The tissue sections weredehydrated using a Leica autostainer by washing once for 2 min in 70%EtOH, twice for 2 min in 95% EtOH, and three times for 2 min in 70%EtOH, then cleared by washing three times for 5 min in xylene. Thesections were permanently mounted with permount to the slide, coveredwith a coverslip, and transferred to a chemical hood to dry.

Example 11 Design of Phase I Clinical Study on Anti-PD-L1 Antibody

Study Design

A Phase I study was conducted to assess the safety and tolerability ofBMS-936559 (also referred to herein and in U.S. Pat. No. 7,943,743 as12A4) in patients with selected advanced solid tumors. Secondaryobjectives included initial assessment of the antitumor activity ofBMS-936559 and pharmacokinetic evaluation.

Pharmacodynamic measures were included under exploratory objectives.Patients were treated in 6-week cycles of BMS-936559 administered as a60-minute intravenous infusion every 2 weeks on days 1, 15, and 29 ofeach cycle. Patients continued treatment for up to 16 cycles unless theyexperienced unacceptable toxicity, disease progression, or withdrewconsent. In some patients who were clinically stable, treatment beyondinitial disease progression was permitted until further progression wasconfirmed.

Dose Escalation

Patients with advanced NSCLC, MEL, CRC, RCC, ovarian (OV), gastric (GC),breast (BC), and pancreatic (PC) carcinoma were eligible to enroll.Using an accelerated titration design, safety was assessed at doses of0.3, 1, 3, and 10 mg/kg. One patient was enrolled in each successivecohort until there was a ≥grade 2 drug-related AE during cycle 1. Twoadditional patients were then enrolled at that dose level and the studywas transitioned to a standard 3+3 design. Intra-patient dose escalationor de-escalation was not permitted. The maximum tolerated dose (MTD) wasdefined as the highest dose where less than one-third of patients had adose-limiting toxicity.

Cohort Expansion

Initially, 5 expansion cohorts (n=16/cohort) were enrolled at 10 mg/kgfor patients with NSCLC, MEL, RCC, OV, and CRC. Based on initial signalsof activity, additional expansion cohorts (up to n=16/cohort) wereenrolled for MEL (at 1.0 and 3.0 mg/kg), NSCLC (squamous or nonsquamoushistology cohorts randomized to 1, 3, or 10 mg/kg), and at 10 mg/kg forPC, BC, and GC.

Patients

Patients were required to have documented advanced NSCLC, MEL, RCC, OV,

CRC, PC, GC, or BC, and have failed at least one prior tumor-appropriatetherapy for advanced/metastatic disease (except for PC or GC patientswho could be treatment-naïve). Other inclusion criteria included age ≥18years, life expectancy ≥12 weeks, Eastern Cooperative Oncology Groupperformance status of ≤2, measurable disease as defined by RECIST v1.0,and adequate hematologic, hepatic, and renal function. Patients withtreated brain metastases were allowed, if stable for at least 8 weeks.Major exclusion criteria included a history of autoimmune disease orother diseases requiring systemic steroids or immunosuppressivemedication, prior therapy with T cell-modulating Abs (includinganti-PD-1, anti-PD-L1 and anti-CTLA-4), history of HIV, or activehepatitis B or C.

In this ongoing study, 207 patients with NSCLC (n=75), MEL (n=55), CRC(n=18), RCC (n=17), OV (n=17), PC (n=14), GC (n=7), or BC (n=4) weretreated with BMS-936559 during a 34-month period and are included in thesafety data. Efficacy was characterized in 160 response-evaluablepatients. The baseline demographic characteristics of the total andresponse-evaluable patient populations were very similar (Brahmer etal., 2012). Among treated patients, 86% had received prior chemotherapyand 28% immunologic or biological therapy. Prior therapies by tumor typeincluded immunotherapy (56%) and B-RAF inhibitor (9%) in patients withMEL; platinum-based chemotherapy (95%) and tyrosine kinase inhibitors(TKI; 41%) in patients with NSCLC; and nephrectomy (94%),anti-angiogenic therapy (82%), and immunotherapy (41%) in patients withRCC (see Brahmer et al., 2012) for more details on patientpre-treatments).

Statistical Analysis

All 207 patients commencing treatment as of the date of analysis wereused for summaries of baseline characteristics and AEs. The efficacypopulation consisted of 160 response-evaluable patients who initiatedtreatment at least 7 months before the date of analysis. AEs were codedusing MedDRA v14.1. Individual best overall responses were derived fromradiographic scan measurements according to modified RECIST v1.0. ORswere confirmed by at least one sequential tumor assessment. Additionaldetails regarding statistical methods are provided in Brahmer et al.,2012.

Example 12

Safety Evaluations on Patients Treated with Anti-PD-L1 Antibody

Safety evaluations (clinical examination and laboratory assessments)were conducted on all treated patients at baseline and regular intervals(weekly during cycle 1 and biweekly thereafter). AE severity was gradedbased on the NCI CTCAE, v3.0. Disease assessment via computed tomography(CT) scans or magnetic resonance imaging was performed at baseline andprior to each treatment cycle.

A MTD was not reached up to the highest tested dose of 10 mg/kg ofBMS-936559. The median duration of therapy was 12 weeks (range 2.0-111.1weeks). A relative dose intensity of ≥90% was achieved in 86% ofpatients. Twelve of 207 patients (6%) discontinued treatment due to aBMS-936559-related AE (see Brahmer et al., 2012, for details).

AEs regardless of causality (any grade) were reported in 188 of 207patients. Investigator-assessed BMS-936559-related AEs were noted in 126of 207 (61%) patients. The most common drug-related AEs were fatigue,infusion reactions, diarrhea, arthralgia, rash, nausea, pruritus, andheadache. Most events were low grade with BMS-936559-related grade 3-4events noted in 19 of 207 (9%) patients (Brahmer et al., 2012). Thespectrum, frequency, and severity of BMS-936559-related AEs were similaracross the dose levels, with the exception of infusion reactions.Drug-related AEOSIs, with potential immune-related etiologies, wereobserved in 81 of 207 (39%) of the patients and included rash,hypothyroidism, hepatitis, and single cases each of sarcoidosis,endophthalmitis, diabetes mellitus, and myasthenia gravis (Brahmer etal., 2012). These AEs were predominantly grade 1-2 and generallyreversible with treatment interruption or discontinuation. Notably, 9patients were treated with corticosteroids for the management of AEs.AEs improved or resolved in all patients. Furthermore, 4 of these 9patients maintained disease control despite treatment withcorticosteroids. Endocrine AEs were managed with replacement therapy andpatients reinitiated treatment with BMS-936559 at the discretion of thetreating physician. Infusion reactions were observed in 21 of 207 (10%)patients, predominantly at 10 mg/kg. They were grade 1-2 with theexception of one grade 3 event at 10 mg/kg. Infusion reactions weregenerally rapidly reversible with antihistamines and antipyretics and,in some cases, corticosteroids. A prophylactic regimen withantihistamines and antipyretics was implemented during the study.Patients with grade 1-2 infusion reactions were able to continuetreatment with BMS-936559 with prophylactic antihistamines andantipyretics and at a reduced infusion rate. BMS-936559-related seriousAEs occurred in 11 of 207 (5%) patients. As of the data analysis date,45 patients (22%) had died (Brahmer et al., 2012); no drug-relateddeaths were observed.

Example 13 Pharmacokinetics/Pharmacodynamics Analyses on Anti-PD-L1Antibody

For PK analyses, serial blood samples were collected and serumconcentrations of BMS-936559 were quantified by ELISA. Peripheral bloodmononuclear cells were isolated from patients at baseline and followingone treatment cycle to assay PD-L1 RO by BMS-936559 on circulatingCD3-positive T-cells via flow cytometry (Brahmer et al., 2010).

Serum concentrations of BMS-936559 increased in a dose-dependent mannerfrom 1-10 mg/kg (n=131). Geometric mean area under the curve (0-14 days)for the 1, 3, and 10 mg/kg dose levels were 2210, 7750, and 36620μg/mL·hr (coefficient of variation [CV] 34-59%), respectively; geometricmean peak concentrations at these dose levels were 27, 83, and 272 μg/mL(CV 30-34%), respectively, after the first dose. The half-life ofBMS-936559 was estimated from population pharmacokinetic data asapproximately 15 days. PD-L1 RO on CD3-positive peripheral bloodlymphocytes was assessed in 29 MEL patients at the end of 1 cycle oftreatment, at BMS-936559 doses from 1-10 mg/kg. Median RO exceeded 65%for all groups (Brahmer et al., 2012).

Example 14 Antitumor Efficacy Exhibited by Anti-PD-L1 Antibody

One-hundred and sixty patients out of the 207 treated were evaluable forresponse and included those with NSCLC, MEL, CRC, RCC, OV, and PC, butnot patients with GC or BC. Clinical activity was observed at all doses≥1 mg/kg (Brahmer et al., 2012). ORs (confirmed complete [CR] or partial[PR] responses) were observed in patients with MEL, NSCLC, RCC, and OV(Table 7), as illustrated by representative spider plots and CT scans(FIGS. 10-13), and many ORs were also durable (Table 8). Four additionalpatients had a persistent reduction in target lesions in the presence ofnew lesions, consistent with an “immune-related” pattern of response;however, for the purpose of calculating response rates, they were notcategorized as responders. Antitumor responses and/or prolonged stabledisease (SD) were observed in patients with a variety of prior therapiesreceived. ORs were observed even in patients with an extensive burden ofmetastatic disease.

In patients with MEL, there were 9 ORs across the 1, 3, and 10 mg/kgdose levels, with response rates of 6%, 29%, and 19%, respectively.Three MEL patients achieved a CR. All 9 MEL patients who experienced anOR started treatment ≥1 year prior to data analysis; of these 5 had aresponse duration ≥1 year. Additionally 14 MEL patients (27%) had SDlasting ≥24 weeks. In patients with NSCLC, there were 5 ORs amongst the3 and 10 mg/kg dose levels, with response rates of 8% and 16%,respectively. There were ORs in patients with non-squamous (n=4) orsquamous histology (n=1). All 5 NSCLC responders started treatment ≥24weeks prior to data analysis; of these, 3 had responses lasting ≥24weeks. Six additional NSCLC patients had SD lasting ≥24 weeks. There was1 PR out of 17 patients with OV (6% response rate) and 3 patients (18%)with SD lasting ≥24 weeks, all at the 10 mg/kg dose. In patients withRCC, there were ORs in 2 of 17 (12%) patients treated at 10 mg/kg withresponses lasting 4 and 18 months, respectively. Seven additional RCCpatients had SD lasting ≥24 weeks.

TABLE 7 Clinical Activity of BMS-936559 in 160 Patients, ResponseEvaluable* ORR^(§) SD ≥24 wk PFSR** at Dose No. of patients (%) No. ofpatients (%) 24 wk (%) Tumor Type (mg/kg) n [95% CI] [95% CI] [95% CI]MEL 0.3 1 0 [0-98]  0 [0-98] N/A 1 18 1 (6) [0.1-27]  6 (33) [13-59] 39[16-61] 3 17 5 (29)^(†) [10-56]  3 (18) [4-43] 47 [21-72] 10 16 3(19)^(††) [4-46]  5 (31) [11-59] 44 [19-68] ALL MEL 52 9 (17) (8-30) 14(27) [16-41] 42 [28-56] NSCLC^(§) 1 11 0 [0-29]  0 [0-29] N/A Squamous 11 0 [0-98]  0 [0-98] N/A Non-Squamous 1 10 0 [0-31]  0 [0-31] N/A 3 13 1(8) [0.2-36]  1 (8) [0.2-36] 34 [7-60] Squamous 3 4 0 [0-60]  1 (25)[0.6-81] 50 [1-99] Non-Squamous 3 9 1 (11) [0.3-48]  0 [0-34] 25 [0-55]10 25 4 (16) [5-36]  5 (20) [7-41] 46 [25-67] Squamous 10 8 1 (13)[0.3-53]  2 (25) [3-65] 47 [10-83] Non-Squamous 10 17 3 (18) [4-43]  3(18) [4-43] 46 [20-72] ALL NCSLC 49 5 (10) [3-22]  6 (12) [5-25] 31[17-45] ALL Squamous 13 1 (8) [0.2-36]  3 (23.1) [5-54] 43 [15-71] ALLNon- 36 4 (11) [3-26]  3 (8) [2-23] 26 [10-42] Squamous OV 3 1 0 [0-98] 0 [0-98] N/A 10 16 1 (6) [0.2-30]  3 (19) [4-46] 25 [4-46] ALL OV 17 1(6) [0.1-29]  3 (18) [4-43] 22 [2-43] RCC 10 17 2 (12) [2-36]  7 (41)[18-67] 53 [29-77] CI denotes Confidence intervals, MEL melanoma, RCCrenal cell carcinoma, NSCLC non-small cell lung cancer, OV ovariancancer, RCC renal cell carcinoma, N/A not applicable, ORR objectiveresponse rate (complete response + partial response), SD stable disease,and PFSR progression-free survival rate. *Efficacy population consistsof response-evaluable patients who initiated treatment at least 7 monthsprior to the date of analysis and had measurable disease at a baselinetumor assessment and at least one of the following: an on-study tumorassessment, clinical progression, or death. ^(†)Includes two CRs.^(††)Includes one CR. ^(§)Objective response rates ({[CR + PR] ÷ n} ×100) are based on confirmed responses only, with confidence intervalscalculated using the Clopper-Pearson method. **Progression-free survivalrate was the proportion of patients who did not progress, and were aliveat 24 weeks, calculated by the Kaplan-Meier methodology, with confidenceintervals using the Greenwood methodIndividual patient responses were adjudicated per RECIST v1.0 withmodification (see study protocol in Brahmer et al. (2012) N. Engl. J.Med. (submitted) for additional information).

TABLE 8 Duration of Objective Responses to BMS-936559* No. of Tumor DosePatients Duration of Response Type (mg/kg) with OR (months)^(†) MEL 1 16.9 3 5 23.5+, 22.9+, 16.2+, 4.1+, 3.5 10 4 24.6+, 23.9+, 18.0+, 17.0NSCLC 1 0 9.2+ 3 1 2.3+ 10 4 16.6+, 12.6+, 9.8, 3.5 RCC 10 2 17, 4 OV 101 1.3+ *MEL denotes melanoma, NSCLC non-small cell lung cancer, RCCrenal cell cancer, OV ovarian cancer. ^(†)Time from first response totime of documented progression, death, or for censored data Denoted by+), time to last tumor assessment.

Sequence Listing Summary SEQ ID NO: Description 1 V_(H) amino acidsequence of 17D8 2 V_(H) amino acid sequence of 2D3 3 V_(H) amino acidsequence of 4H1 4 V_(H) amino acid sequence of 5C4 5 V_(H) amino acidsequence of 4A11 6 V_(H) amino acid sequence of 7D3 7 V_(H) amino acidsequence of 5F4 8 V_(L) amino acid sequence of 17D8 9 V_(L) amino acidsequence of 2D3 10 V_(L) amino acid sequence of 4H1 11 V_(L) amino acidsequence of 5C4 12 V_(L) amino acid sequence of 4A11 13 V_(L) amino acidsequence of 7D3 14 V_(L) amino acid sequence of 5F4 15 V_(H) amino acidsequence of 3G10 16 V_(H) amino acid sequence of 12A4 17 V_(H) aminoacid sequence of 10A5 18 V_(H) amino acid sequence of 5F8 19 V_(H) aminoacid sequence of 10H10 20 V_(H) amino acid sequence of 1B12 21 V_(H)amino acid sequence of 7H1 22 V_(H) amino acid sequence of 11E6 23 V_(H)amino acid sequence of 12B7 24 V_(H) amino acid sequence of 13G4 25V_(L) amino acid sequence of 3G10 26 V_(L) amino acid sequence of 12A427 V_(L) amino acid sequence of 10A5 28 V_(L) amino acid sequence of 5F829 V_(L) amino acid sequence of 10H10 30 V_(L) amino acid sequence of1B12 31 V_(L) amino acid sequence of 7H1 32 V_(L) amino acid sequence of11E6 33 V_(L) amino acid sequence of 12B7 34 V_(L) amino acid sequenceof 13G4 35 V_(H) amino acid sequence of 28-8 36 V_(L) amino acidsequence of 28-8

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1-17. (canceled)
 18. A method of treating a late stage non-small cell lung cancer (NSCLC) tumor in a human subject, comprising administering to the subject about 10 mg/kg of an anti-PD-L1 antibody every 2 weeks, wherein the anti-PD-L1 antibody is administered intravenously over 60 minutes infusion; and wherein the subject is pretreated for a chemotherapy and a radiotherapy.
 19. The method of claim 18, wherein the tumor is Stage IV NSCLC.
 20. The method of claim 18, wherein the tumor is advanced or recurrent.
 21. The method of claim 18, wherein the tumor is metastatic.
 22. The method of claim 18, wherein the subject has previously had a stage I and/or II tumor that has surgically been removed.
 23. The method of claim 18, wherein the treatment lasts more than 12 weeks.
 24. The method of claim 18, wherein the anti-PD-L1 antibody is formulated in a pharmaceutical composition.
 25. The method of claim 24, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable salt, an anti-oxidant, an aqueous carrier, a non-aqueous carrier, an adjuvant, or any combination thereof.
 26. The method of claim 25, wherein the adjuvant comprises a preservative, a wetting agent, an emulsifying agent, a dispersing agent, or any combination thereof.
 27. The method of claim 25, wherein the salt comprises a sodium salt.
 28. The method of claim 25, wherein the salt comprises sodium chloride.
 29. The method of claim 18, wherein the subject is PD-L1 positive.
 30. The method of claim 18, wherein the subject is PD-L1 negative.
 31. The method of claim 18, further comprising administering an anti-cancer agent.
 32. The method of claim 19, further comprising administering an anti-cancer agent.
 33. The method of claim 20, further comprising administering an anti-cancer agent.
 34. The method of claim 21, further comprising administering an anti-cancer agent.
 35. The method of claim 22, further comprising administering an anti-cancer agent.
 36. The method of claim 23, further comprising administering an anti-cancer agent.
 37. The method of claim 31, wherein the anti-cancer agent is an anti-CTLA-4 antibody.
 38. The method of claim 32, wherein the anti-cancer agent is an anti-CTLA-4 antibody.
 39. The method of claim 33, wherein the anti-cancer agent is an anti-CTLA-4 antibody.
 40. The method of claim 34, wherein the anti-cancer agent is an anti-CTLA-4 antibody.
 41. The method of claim 35, wherein the anti-cancer agent is an anti-CTLA-4 antibody.
 42. The method of claim 36, wherein the anti-cancer agent is an anti-CTLA-4 antibody.
 43. The method of claim 18, wherein at least 1% of tumor cells exhibit membrane PD-L1.
 44. The method of claim 18, wherein at least 5% of tumor cells exhibit membrane PD-L1.
 45. The method of claim 18, further comprising measuring membranous PD-L1 on tumor cells.
 46. The method of claim 45, wherein the measuring comprises an immunohistochemistry.
 47. The method of claim 46, wherein the immunohistochemistry is performed using an antibody comprising: (a) a heavy chain (HC) complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in the HC-CDR1 of SEQ ID NO: 35; (b) an HC-CDR2 comprising the amino acid sequence set forth in the HC-CDR2 of SEQ ID NO: 35; (c) an HC-CDR3 comprising the amino acid sequence set forth in the HC-CDR3 of SEQ ID NO: 35; (d) a light chain (LC) CDR1 comprising the amino acid sequence set forth in the LC-CDR1 of SEQ ID NO: 36; (e) an LC-CDR2 comprising the amino acid sequence set forth in the LC-CDR2 of SEQ ID NO: 36; and (f) an LC-CDR1 comprising the amino acid sequence set forth in the LC-CDR1 of SEQ ID NO:
 36. 